Compositions and methods for treatment of edema

ABSTRACT

Provided are pharmaceutical compositions and methods of treating or preventing edema, using an anti-T cell agent, an anti-TGF-β1 agent, or an anti-angiotensin agent, preferably a combination of at least two such agents. The pharmaceutical compositions can be formulated for systemic or local administration, and are preferably administered topically.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/549,156, filed on Aug. 5, 2017, now patented, which is the nationalstage under 35 U.S.C. 371 of International Application No.PCT/US2016/016680, filed on Feb. 5, 2016, now expired, which claims thebenefit of priority of U.S. Provisional Patent Application No.62/112,273, filed on Feb. 5, 2015, the entire contents of all of whichare hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersHL111130 and CA008748, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

COPYRIGHT

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

INCORPORATION BY REFERENCE

For countries that permit incorporation by reference, all of thereferences cited in this disclosure are hereby incorporated by referencein their entireties. In addition, any manufacturers' instructions orcatalogues for any products cited or mentioned herein are incorporatedby reference. Documents incorporated by reference into this text, or anyteachings therein, can be used in the practice of the present invention.Documents incorporated by reference into this text are not admitted tobe prior art.

BACKGROUND

Lymphedema is a chronic debilitating disease that in the United Statesand Western countries occurs most frequently as a complication of cancertreatment. In this setting, lymphedema occurs as a result of iatrogenicinjury to the lymphatic system most commonly after lymph node dissectionbut also as a result of wide skin excisions and adjuvant therapy withradiation. Purushotham et al., J. Clin. Oncol. 23:4312-4321 (2005);Szuba et al., Cancer 95:2260-2267 (2002); Tsai et al., Ann. Surg. Oncol.16:1959-72 (2009). It is estimated that as many as 1 in 3 patients whoundergo lymph node dissection will go on to develop lymphedema andconservative estimates suggest that as many as 50,000 new patients arediagnosed annually. DiSipio et al., Lancet Oncol. 14:500-515 (2013);Petrek et al., Cancer 83:2776-2781 (1998). Because lymphedema is alife-long disease with no cure, the number of affected individuals isincreasing annually with current estimates ranging between 5-6 millionAmericans (Rockson et al., Ann. NY Acad. Sci. 1131:147-154 (2008)) andover 200 million people world-wide. It is likely that this number willcontinue to increase in the future since the development of lymphedemais nearly linearly related with cancer survivorship, and because theprevalence of known risk factors for lymphedema, such as obesity andradiation, is rising. Erickson et al., J. Natl. Cancer Inst. 93:96-111(2001).

Lymphedema is disfiguring and debilitating; patients have chronicswelling of the affected extremity, recurrent infections, limitedmobility, and decreased quality of life. Hayes et al., Cancer118:2237-2249 (2012). In addition, once lymphedema develops it isusually progressive. Despite the fact that lymphedema is common andhighly morbid, there is currently no cure, and treatment is palliativewith a goal of preventing disease progression rather than restoration oflymphatic function. Velanovich et al., Am. J. Surg. 177:184-187 (1999);Beaulac et al., Arch. Surg. 137; 1253-1257 (2002). As a result, patientsare required to wear tight, uncomfortable garments for the rest of theirlives, in an effort to prevent lymphatic fluid buildup in the affectedextremity, and to undergo intense and time consuming physical therapytreatments. Koul et al., Int. J. Radiat. Oncol. Biol. Phys., 67:841-846(2007). In addition, despite on-going chronic care, some patients stillhave severe progression of their disease with increasing swelling andfrequent infections in the lymphedematous limb. Currently there is noknown pharmacologic therapy that can halt progression or promoteresolution of lymphedema. Cormier et al., Ann. Surg. Oncol. 19:642-651(2012). Development of targeted treatments for lymphedema is thereforean important goal and is an unmet biomedical need.

Recent studies have shown that fibrosis is not only a clinical hallmarkof lymphedema, but also a key pathologic regulator of the disease.Cheville et al., Semin. Radiat. Oncol. 13:214-225 (2003); Mihara et al.,PLoS One 7:e41126 (2012); Rasmussen et al., Curr. Opin. Biotechnol.20:74-82 (2009). Transforming growth factor beta-1 (TGF-β1) is acritical regulator of fibrosis in a variety of organ systems, acting viadirect mechanisms to increase collagen production by fibroblasts anddecrease turnover of matrix products. Willis et al., Am. J. Pathol.166:1321-1332 (2005); Sakai et al., Am. J. Pathol. 184:2611-2617 (2014);Qi et al., Am. J. Physiol. Renal Physiol. 288:F800-F809 (2005); Bonniaudet al., J. Immunol. 173:2099-2108 (2004); Fujimoto et al., Biochem.Biophys. Res. Commun. 305:1002-1007 (2003); Stramer et al., J. CellPhysiol. 203:226-232 (2005); Kawakami et al., J. Invest. Dermatol.110:47-51 (1998); Li et al., Circulation 96:874-881 (1997); Martinez etal., Hepatology 21:113-119 (1995); Peltonen et al., J. Invest. Dermatol.97:240-248 (1991); Van Laethem et al., Gastroenterology 110:576-582(1996). In addition, TGF-β1 is a key regulator of inflammatory responsesand is thought to regulate fibrosis indirectly by modulating chronicinflammation. Pesce et al., PLoS Pathog. 5:e1000371 (2009). We haverecently shown that the expression of TGF-β1 is markedly increased inlymphedematous tissues, both clinically and in mouse models oflymphedema. Inhibition of TGF-β1 using immunotherapy significantlyaccelerates lymphatic regeneration, decreases fibrosis, decreasesinflammation, and improves lymphatic function in the mouse tail model.Avraham et al., Plast. Reconstr. Surg. 124:438-450 (2009); Clavin etal., Am. J. Physiol. Heart Circ. Physiol. 295:H2113-H2127 (2008);Avraham et al., Am. J, Pathol. 177:3202-3214 (2010).

Inhibition of fibrotic responses preserves the capacity of the lymphaticsystem to transport interstitial fluid and inflammatory cells. Recentstudies from our lab have shown that CD4⁺ cells play a crucial role inthe regulation of fibrosis in both clinical and animal models oflymphedema. Avraham et al., Am. J. Pathol. 177:3202-3214 (2010); Avrahamet al., FASEB J. 27:1114-1126 (2013); Zampell et al., Am. J. Physiol.Cell Physiol. 302:C392-C404 (2012); Zampell et al., PLoS ONE 7:e49940(2012). For example, we have found that clinical lymphedema biopsyspecimens and animal models of lymphedema are infiltrated by CD4⁺ cells,and that the number of these cells correlates with the degree offibrosis and clinical severity of disease. Avraham et al., FASEB J.27:1114-1126 (2013). Patients with late stage lymphedema hadsignificantly more infiltrating T cells in general, specifically moreCD4⁺ cells, than those with early stage disease. Improvements inclinical symptoms of lymphedema after lymphovenous bypass, a procedurein which obstructed lymphatics are shunted to the venous circulation, isassociated with decreased tissue fibrosis and decreased CD4⁺ cellinfiltration. Torrisi, et al., Lymphat. Res. Biol. 13:46-53 (2015).

The CD4⁺ cell response in lymphedema, similar to otherfibroproliferative disorders, is characterized by a mixed Th1/Th2 cellpopulation. Avraham et al., FASEB J. 27:1114-1126 (2013). Naïve CD4+ Tcells, also known as T-helper or Th cells, patrol secondary lymphoidstructures and, upon activation, differentiate along numerousdistinct/overlapping cell types (e.g., Th1, Th2, Th17, T regulatory,etc.). The Th2 subset of cells plays a key role in regulation ofresponses to parasites and some autoimmune responses. These cells havealso been implicated in the pathology of fibroproliferative diseases ina number of organ systems including the heart, lung, kidneys and skin.More recent studies have shown that the number of Th2 is increased intissue biopsies obtained from patients with lymphedema and thatinhibition of Th2 differentiation decreases the pathology of lymphedemain mouse models.

Depletion of CD4⁺ cells (but not other inflammatory cell types includingCD8⁺ cells or macrophages) or inhibition of Th2 differentiation (but notgeneralized inflammation or inhibition of interleukin-6) markedlydecreases the degree of fibrosis, increases lymphangiogenesis andlymphatic fluid transport, and effectively treats established lymphedemain preclinical mouse models. Avraham et al., FASEB J. 27:1114-1126(2013); Zampell et al., PLoS ONE 7:e49940 (2012); Ghanta et al., Am. J.Physiol. Heart Circ. Physiol. 308:H1065-1077 (2015). These findings aresupported by recent studies demonstrating that T cells potently inhibitlymphangiogenesis by producing anti-lymphangiogenic cytokines/growthfactors, including interferon gamma (IFN-γ), interleukin (IL)-4, IL-13,and TGF-β1. Kataru et al., Immunity 34:96-107 (2011); Shin et al., Nat.Commun. 6:6196 (2015); Shao et al., J. Interferon. Cytokine Res.26:568-574 (2006); Oka et al., Blood 111:4571-4579 (2008). Takentogether, these findings suggest that infiltrating CD4⁺ cells inlymphedematous tissues decrease lymphatic function through multiplemechanisms including induction of structural changes of lymphaticvessels secondary to tissue fibrosis and inhibition of collaterallymphatic vessel formation.

Previous experimental treatments for lymphedema have focused on deliveryof lymphangiogenic cytokines. Skobe et al., Nat. Med. 7:192-198 (2001).For example, some previous studies have focused on repairing damagedlymphatics using lymphangiogenic cytokines such as vascular endothelialgrowth factor-c (VEGF-C). Tammela et al., Nat. Med. 13:1458-1466 (2007);Baker et al., Breast Cancer Res. 12:R70 (2010). Although promising,application of this approach, particularly to cancer patients, may beuntenable as these same mechanisms regulate tumor growth and metastasis,raising the risk of cancer metastases or recurrence. Zhang et al.,Cancer Res. 70:2495-2503 (2010); Yu et al., J. Exp. Clin. Cancer Res.28:98 (2009); Sugiura et al., Int. J. Oncol. 34:673-680 (2009); Gu etal., Clin. Exp. Metastasis 25:717-725 (2008); Kazama et al.,Hepatogastroenterology 54:71-76 (2007); Hirakawa et al., Blood109:1010-1017 (2007). In contrast, depletion of CD4+ T cells locally cantreat the underlying pathology rather than only promotinglymphangiogenesis, and can therefore be much safer for use in cancerpatients. This approach can thus enable treatment of cancer survivorsduring flare-ups/exacerbations of lymphedema, add to conservativetherapy in non-surgical patients, prevent disease development in highrisk patients, or improve outcomes of surgical treatments forlymphedema.

Tacrolimus is an anti-T cell agent that is FDA approved as a topicalformulation and used to treat cutaneous inflammatory/fibrotic diseasesincluding atopic dermatitis (Ruzicka et al., N. Engl. J. Med.337:816-821 (1997)), psoriasis (Wang et al., J. Cutan. Med. Surg.18:8-14 (2014)), and localized scleroderma (Mancuso et al., Br. JDermatol. 152:180-182 (2005)). Tacrolimus is a macrolide produced by thesoil bacterium Streptomyces tsukubaensis that is well-tolerated whenused for prevention of transplant rejection and treatment of a varietyof autoimmune diseases. It exerts its anti-T cell properties by bindingto FK-506 binding protein 12 (FKBP-12) thus inhibiting calcineurin, andultimately decreasing IL-2 expression. Clipstone et al., Nature357:695-697 (1992). Because IL-2 is essential for T cell activation anddifferentiation of CD4⁺ T cells, calcineurin inhibitors have profoundCD4⁺ cell immunosuppressive effects. Liao et al., Immunity 38:13-25(2013); Rautajoki et al., Ann. Med. 40:322-335 (2008).

Teriflunomide is an immunosuppressive agent that decreases T cellinflammatory responses. Oral administration of teriflunomide isFDA-approved for the treatment of multiple sclerosis. Williamson et al.,J. Biol. Chem. 270:22467-22472 (1995); Davis et al., Biochem.35:1270-1273 (1996); Iglesias-Bregna et al., J. Pharmacol. Exp. Ther.347:203-211 (2013). Teriflunomide is the active metabolite ofleflunomide, and inhibits de novo pyrimidine synthesis by blocking theenzyme dihydroorotate dehydrogenase. Teriflunomide has also been shownto inhibit activation of Signal transducer and activator oftranscription-6 (STAT-6) a key regulator of Th2 differentiation. Olsanet al., Proc. Natl. Acad. Sci. USA 108:18067-18072 (2011). As a resultof these mechanisms, teriflunomide inhibits actively dividing Th2 cellsand decreases inflammatory responses.

Pirfenidone is a compound that has anti-fibrotic and anti-inflammatoryeffects.

Recent studies have suggested that this activity is due, at least inpart, to inhibition of production and activity of TGF-β. Iyer et al., J.Pharmacol. Exp. Ther. 291:367-373 (1999); Tada et al., Clin. Exper.Pharmacol. Physiol. 28:522-527 (2001); Oku et al., Eur. J. Pharmacol.590:400-408 (2008); Tian et al., Chin. Med. Sci. J. 21:145-151 (2006);Schaefer et al., Eur. Respir. Rev. 20:85-97 (2011). It is currentlyapproved in the United States by the FDA for oral administration in thetreatment of idiopathic pulmonary fibrosis (IPF) after its safety andefficacy were established in three clinical trials of 1,247 patientswith IPF. Taniguchi et al., Eur. Respir. J. 35:821-829 (2010); Noble etal., Lancet 377:1760-1769 (2011); King et al., N. Engl. J. Med.370:2083-2092 (2014). In addition to the treatment of IPF, pirfenidonehas been clinically evaluated for its safety and efficacy for thetreatment of other chronic fibrotic disorders, including renal fibrosis,hepatic fibrosis, and myelofibrosis. Tada et al., Clin. Exper.Pharmacol. Physiol. 28:522-527 (2001); Cho et al., Clin. J. Am. Soc.Nephrol. 2:906-913 (2007); Nagai et al., Intern. Med. 41:1118-1123(2002); Raghu et al., Am. J. Respir. Crit. Care Med. 159:1061-1069(1999); Gahl et al., Mol. Genet. Metab. 76:234-242 (2002);Armendariz-Borunda et al., Gut 55:1663-1665 (2006); Angulo et al., Dig.Dis. Sci. 47:157-161 (2002); Mesa et al., Brit. J. Haematol. 114:111-113(2001).

Captopril is an angiotensin-converting enzyme (ACE) inhibitor, approvedby the FDA for oral administration in the treatment of hypertension andcertain types of heart failure and diabetic nephropathy. ACE convertsangiotensin I (AngI) to angiotensin II (AngII) and causes blood vesselconstriction, inhibits vasodilatation, and indirectly regulatesintravascular fluid volumes by effects on the renin-angiotensin-system(RAS). Therefore, inhibition of ACE has been a mainstay therapy forhypertension. More recent studies have shown that AngII is also a keyregulator of fibrosis in a variety of organ systems, including thekidney, liver, and lung. Langham et al., Diabetes Care 29:2670-2675(2006); Alves de Albuquerque et al., Kidney Intl. 65:846-859 (2004);Osterreicher et al., Hepatology. 50:929-938 (2009); Mak et al., Mol.Ther. 23:1434-1443 (2015); Wang et al., Cell Physiol. Biochem.36:697-711 (2015). The pro-fibrotic effects of AngII are mediated by anumber of mechanisms, including production of reactive oxygen species,production of chemokines and cytokines, increased expression of adhesionmolecules, and regulation of TGF-β expression/activity. In contrast,AngI has anti-proliferative and anti-fibrotic activities by activatingits cell surface receptor, Mas. Clarke et al., Int. J. Hypertens.2012:307315 (2011). As a result, inhibitors of ACE and/or AngII, such ascaptopril, losartan, and other similar medications, have been proposedas a potential therapeutic option for fibrotic disorders of the lung,kidney, and liver.

There are currently no pharmacologic therapies available for thetreatment of lymphedema. Previous studies on lymphedema have focused ontreatment with systemic medications. For example, coumarin taken bymouth has been used in patients with lymphedema with modest success.Casley-Smith et al., BMJ 307:1037-1041 (1993); Casley-Smith et al., N.Engl. J. Med. 329:1158-1163 (1993); Casley-Smith et al., Australas J.Dermatol. 33:69-74 (1992); Loprinzi et al., N. Engl. J. Med. 340:346-350(1999). However, widespread clinical application of this drug has beenhampered by significant toxicity including liver failure and death.Loprinzi et al., N. Engl. J. Med. 340:346-350 (1999). Strategiestargeting fibrosis—in particular, inhibition of generalized CD4⁺inflammatory responses, Th2 differentiation, and/or the TGF-βpathway—hold clinical promise for treating lymphedema. Although highlyeffective, systemic depletion of CD4+ cells is not clinically viable dueto unacceptable morbidity and systemic complications such as infections,cancer recurrence, and autoimmune disorders. In contrast, local deliveryof agents to treat pathologic events related to lymphedema is a novelapproach that may limit systemic toxicity. Because lymphedema isprimarily a disease of the skin and subcutaneous soft tissues of theextremities, it is possible to use topical approaches, which might bebetter-tolerated and provide a more targeted approach, thereby avoidingsystemic complications. Accordingly, there is a need in the art fornovel treatments for lymphedema, especially topical treatments.

SUMMARY OF THE INVENTION

Some of the main aspects of the present invention are summarized below.

Additional aspects are described in the Detailed Description of theInvention, Examples, Drawings, and Claims sections of this disclosure.The description in each section of this disclosure is intended to beread in conjunction with the other sections. Furthermore, the variousembodiments described in each section of this disclosure can be combinedin various different ways, and all such combinations are intended tofall within the scope of the present invention.

In one aspect, the invention provides a pharmaceutical compositioncomprising a combination of anti-T cell, anti-TGF-β1, and/oranti-angiotensin agents. For example, the invention provides apharmaceutical composition comprising: (i) an effective amount of one ormore anti-T cell agents and (ii) an effective amount of one or moreanti-TGF-β1 agents and/or an effective amount of one or moreanti-angiotensin agents. In a particular embodiment, the inventionprovides a pharmaceutical composition comprising (i) an effective amountof one or more anti-T cell agents and (ii) an effective amount of one ormore anti-TGF-β1 agents. In another embodiment, the invention provides apharmaceutical composition comprising: (i) an effective amount of one ormore anti-T cell agents and (ii) an effective amount of one or moreanti-angiotensin agents. In still a further embodiment, the inventionprovides a pharmaceutical composition comprising: (i) an effectiveamount of one or more anti-T cell agents and (ii) an effective amount ofone or more anti-TGF-β1 agents and (iii) an effective amount of one ormore anti-angiotensin agents.

In one embodiment, the anti-T cell agent is selected from the groupconsisting of tacrolimus, teriflunomide, leflunomide, cyclosporine,pimecrolimus, denileukin diftitox, and Basiliximab. In one embodiment,the anti-TGF-β1 agent or anti-angiotensin agent is selected from thegroup consisting of pirfenidone, captopril, zofenopril, enalapril,lisinopril, ramipril, quinapril, perindopril, benazepril, imidapril,trandolapril, cilazapril, fosinopril, losartan, irbesartan, olmesartan,candesartan, telmisartan, valsartan, and fimasartan. In a particularembodiment, the anti-angiotensin agent is an ACE inhibitor, for example,an ACE-2 agonist. The composition can be formulated for systemicadministration or for local administration. In a preferred embodiment,the composition is formulated for topical administration.

The pharmaceutical composition of the invention can comprise anycombination of anti-T cell, anti-TGF-β1, and/or anti-angiotensin agents.For instance, in one embodiment, the composition comprises tacrolimusand pirfenidone. In another embodiment, the composition comprisestacrolimus, pirfenidone, and teriflunomide. In an additional embodiment,the composition comprises tacrolimus, pirfenidone, and leflunomide. Infurther aspects, the composition comprises tacrolimus and captopril; orteriflunomide and captopril; or leflunomide and captopril; orpirfenidone and captopril; or tacrolimus, captopril, and teriflunomide;or tacrolimus, captopril, and leflunomide; or tacrolimus, captopril, andpirfenidone.

In an embodiment in which the pharmaceutical composition is formulatedfor topical administration, the composition can comprise about 0.01% toabout 1% tacrolimus, preferably about 0.05 to about 0.2% tacrolimus;about 0.1 mg/ml to about 5 mg/ml pirfenidone, preferably about 0.5 mg/mlto about 2 mg/ml pirfenidone; about 10 mg/ml to about 50 mg/mlteriflunomide, preferably about 20 mg/ml to about 30 mg/mlteriflunomide; about 1% to about 20% leflunomide, preferably about 5% toabout 15% leflunomide; and/or about 1% to about 20% captopril. Thepharmaceutical composition is preferably in a form selected from anointment, a cream, a lotion, a paste, a gel, a mousse, a foam, alacquer, a suspension, a liquid, and a spray. In a preferred embodiment,the composition is in the form of an ointment.

The pharmaceutical composition of the invention can be for use intreating or preventing edema.

The invention also provides a method of treating or preventing edema,the method comprising administering to a subject in need thereof apharmaceutical composition comprising an effective amount of one or moredrugs selected from the group consisting of tacrolimus, teriflunomide,leflunomide, cyclosporine, pimecrolimus, denileukin diftitox,Basiliximab, pirfenidone, captopril, zofenopril, enalapril, lisinopril,ramipril, quinapril, perindopril, benazepril, imidapril, trandolapril,cilazapril, fosinopril, losartan, irbesartan, olmesartan, candesartan,telmisartan, valsartan, and fimasartan.

In one aspect, the edema is lymphedema.

In one aspect, the method of the invention can comprise administering acombination of anti-T cell, anti-TGF-β1, and/or anti-angiotensin agents.In a particular embodiment, the method comprises administering apharmaceutical composition comprising: (i) an effective amount of one ormore anti-T cell agents selected from the group consisting oftacrolimus, teriflunomide, leflunomide, cyclosporine, pimecrolimus,denileukin diftitox, and Basiliximab; and (ii) an effective amount ofone or more anti-TGF-β1 agents and/or anti-angiotensin agents selectedfrom the group consisting of pirfenidone, captopril, zofenopril,enalapril, lisinopril, ramipril, quinapril, perindopril, benazepril,imidapril, trandolapril, cilazapril, fosinopril, losartan, irbesartan,olmesartan, candesartan, telmisartan, valsartan, and fimasartan. Themethod can comprise administering any combination of anti-T cell,anti-TGF-β1, and/or anti-angiotensin agents. For instance, in oneembodiment, the method comprises administering a pharmaceuticalcomposition comprising tacrolimus and pirfenidone. In anotherembodiment, the method comprises administering a pharmaceuticalcomposition comprising tacrolimus, pirfenidone, and teriflunomide. In anadditional embodiment, the method comprises administering apharmaceutical composition comprising tacrolimus, pirfenidone, andleflunomide. In further aspects, the method comprises administering apharmaceutical composition comprising tacrolimus and captopril; orteriflunomide and captopril; or leflunomide and captopril; orpirfenidone and captopril; or tacrolimus, captopril, and teriflunomide;or tacrolimus, captopril, and leflunomide; or tacrolimus, captopril, andpirfenidone.

In the methods of the invention, the pharmaceutical composition can beadministered systemically or locally. In a preferred method, thepharmaceutical composition is administered topically. In this aspect ofthe invention, the pharmaceutical composition about 0.01% to about 1%tacrolimus, preferably about 0.05 to about 0.2% tacrolimus; about 0.1mg/ml to about 5 mg/ml pirfenidone, preferably about 0.5 mg/ml to about2 mg/ml pirfenidone; about 10 mg/ml to about 50 mg/ml teriflunomide,preferably about 20 mg/ml to about 30 mg/ml teriflunomide; about 1% toabout 20% leflunomide, preferably about 5% to about 15% leflunomide;and/or about 1% to about 20% captopril. In a method of topicaladministration, the pharmaceutical composition can be in a form selectedfrom an ointment, a cream, a lotion, a paste, a gel, a mousse, a foam, alacquer, a suspension, a liquid, and a spray. Preferably, thepharmaceutical composition is in the form of an ointment.

It is within the skill of the ordinary artisan to determine a dosingschedule and duration for systemic or local administration. In oneembodiment, the pharmaceutical composition is administered topically atleast once a day. In another embodiment, the pharmaceutical compositionis administered topically at least twice a day. Where the pharmaceuticalcomposition or method involves prevention of edema, particularlyprevention of lymphedema, the composition can be administered withinabout six weeks of a lymphatic injury, preferably within about two weeksof a lymphatic injury.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that topical tacrolimus decreases tail lymphedema. (A)shows representative photographs of mouse tails after surgical excisionof superficial/deep collecting lymphatics and treatment with or withouttopical tacrolimus beginning either 2 weeks (early treatment) or 6 weeks(late treatment) after surgery. Arrows indicate initiation of therapy.(B) is a graphical representation of tail volume changes after early(*p=0.021) or late (*p=0.018) treatment with tacrolimus as compared withcontrols. (C) (upper panel) shows representative cross sectionalhistological images of control and early tacrolimus-treated mouse tailsharvested 6 weeks after lymphatic ablation. Brackets show soft tissuethickness. (C) (lower panel) shows quantification of soft tissue changesafter early or late treatment with tacrolimus (*p=<0.001). (D) showsquantification of whole blood tacrolimus levels demonstratingimmunosuppressive concentrations in systemically-treated animals (4 mgkg⁻¹ IP daily) and non-immunosuppressive levels in the topically—treatedgroup (*p=0.007). (E) shows flow cytometry plots (upper panel) andquantification of blood T cells (lower panel) in animals treated withvehicle control, topical tacrolimus, or systemic tacrolimus. Flow plotsrepresent Side Scatter Area (SSA) on the y-axis and CD3⁺ representing Tcells on the x-axis. Note significant reduction in T cells only insystemically-treated animals (*p=0.012).

FIG. 2 shows that topical tacrolimus decreases inflammation afterlymphatic injury. (A_(i)), (B_(i)), (C_(i)), (D_(i)) show representative40× images of tail tissue sections harvested from control andtacrolimus-treated animals 6 weeks after surgery with immunofluorescentlocalization of CD45⁺ (A_(i)), CD3⁺ cells (B_(i)), CD4⁺ cells (C_(i)),and IFN-γ⁺ cells (D₁). Higher-power (80×) images are shown in the topright corner inset of each figure. Lymphatic vessels are stained(LYVE-1) in each figure. Quantifications of cell counts for both earlyand late treatments are shown to the right of each figure in (A_(ii)),(B_(ii)), (C_(ii)), (D_(ii)) (p<0.001 for all).

FIG. 3 shows that topical tacrolimus decreases fibrosis in lymphedema.(A_(i)) shows representative 40× images of tail tissues harvested fromcontrol or early treatment with tacrolimus 6 weeks after surgery withimmunofluorescent localization of type I collagen and lymphatic vessels.Quantifications of collagen I-staining area in both early and latetreatment are shown to the right in (A_(ii)) (p<0.001 for both). (B_(i))shows representative 40× images of picrosirius red staining of tailtissues harvested from control and early tacrolimus treated animals(collagen I and collagen III deposition). Quantification of Scar Index(red:green ratio) is shown to the right in (B_(ii)) (p=0.036 early;p<0.001 late). (C_(i)) and (D_(i)) show representative 40×immunofluorescent co-localization of TGF-β1 or pSMAD3 with lymphaticvessel in tail tissues harvested from animals treated with control orearly treatment with tacrolimus 6 weeks after surgery. Quantification ofthe number of positive cells/0.25 mm² area for both is shown to theright of each figure in (C_(ii)) and (D_(ii)).

FIG. 4 shows that tacrolimus improves lymphatic function after surgicallymphatic injury. (A) shows representative ICG images of mouse tails 6weeks after surgery following early treatment with or withouttacrolimus. Note flow of ICG proximally across the wound in tacrolimustreated animals. Inset shows photograph of same mice for orientation.(B_(i)) and (B_(ii)) show decay-adjusted uptake of ^(99m)Tc by sacrallymph nodes 6 weeks after surgical ligation of superficial/deeplymphatics in control and early tacrolimus (beginning 2 weeks aftersurgery) treated animals (*p=0.005). Gross photographs of mouse tailsshown in the upper panel of (B_(ii)) are for orientation and show siteof ^(99m)Tc injection and location of sacral lymph nodes; representativeheat maps are shown in the lower panel with the white arrow pointingtoward the sacral lymph nodes. (C) shows representative ICG images ofhind limbs obtained 50 minutes after distal foot injection in micetreated with or without tacrolimus 4 weeks after PLND. White arrows showdermal backflow of ICG. Inset photograph shows orientation. (D_(i)) and(D_(ii)) are graphical representations of lymphatic vessel pulsations inhind limb collecting vessels of mice treated with or without tacrolimus4 weeks after PLND. Quantification of pulsation frequency is shown tothe right (*p=0.001). (E) shows representative 40× fluorescentco-localization images of inflammatory cells (CD45⁺; upper panel), iNOS⁺cells (lower panel) and lymphatic vessels (LYVE-1⁺) in tissues harvestedfrom the distal hind limbs of animals treated with control or tacrolimus4 weeks after PLND. Note peri-lymphatic accumulation of CD45⁻ and iNOS⁺cells. (F) and (G) show quantification of peril-lymphatic CD45⁺ cells((F) and iNOS⁺ cells ((G) in distal hind limb tissues of control ortacrolimus treated animals harvested 4 weeks after PLND.

FIG. 5 shows that topical tacrolimus increases formation of collaterallymphatic vessels after lymphatic injury. (A_(i)) shows representativelongitudinal immunofluorescent 40× images of LYVE-1 vessels bridging thesurgically created tail wounds of control and early treated tacrolimusmice harvested 6 weeks after lymphatic injury; inset shows area wherelongitudinal sections were obtained. (A_(ii)) shows quantification ofbridging lymphatic vessel density (LVD) in the wounded portion of thetail in control versus early or late treated tacrolimus mice (p<0.001for both). (A) shows qPCR of RNA harvested from control and earlytreated tacrolimus mouse tail tissues harvested 6 weeks after lymphaticinjury, demonstrating relative expression of VEGF-C (p=0.264), TGF-β1(p=0.006), and IFN-γ (p=0.014). (B) is a photograph of mouse hind limbshowing site of collateral vessel formation draining towards theinguinal lymph nodes before and after PLND. (C) shows representative ICG(left panels) and 40× immunofluorescent images of LYVE-1⁺ vessels (rightpanel showing area in box) in control and tacrolimus treated animals 4weeks following PLND. (D) shows quantification of collateral lymphaticLVD in the aterolateral thigh region of animals treated with control ortacrolimus (p<0.001).

FIG. 6 shows that tacrolimus increases inflammatory lymphangiogenesis.(A) Representative gross (left panels) and whole mount 5× images (rightpanels) of mouse corneas stained for lymphatic vessels (LYVE-1⁺/weaklyCD31⁺) and blood vessels (CD31⁺/LYVE-1⁻) harvested 2 weeks after sutureplacement and treatment either with vehicle control or systemictacrolimus. Box denotes area shown in whole mount images. (B) and (C)show quantification of corneal lymphatic (LYVE-1⁺) and blood(CD31⁺/LYVE-1⁻) vessels. (D) shows representative florescent whole mount5× images of ear wounds localizing LYVE-1⁺ in control or topicaltacrolimus treated animals harvested 4 weeks after wounding. Insetphotograph shows area where sections were obtained. (E) showsquantification of the LYVE-1⁺ staining area in ear skin (within 400 μmof the wound) demonstrating an increase in lymphangiogenesis intacrolimus treated animals (p<0.001). (F) shows quantification oflymphatic vessel branch-points per unit area in ear wounds treated withor without tacrolimus demonstrating increased branching in tacrolimustreated animals (p<0.001).

FIG. 7 shows that topical tacrolimus does not decrease circulating CD4⁺T cells. Representative flow plots of peripheral blood CD4⁺ cells (upperpanel) with quantification of CD4⁺ T cells (lower panel) are shown after2 week treatment with topical tacrolimus, systemic tacrolimus, orvehicle control.

FIG. 8 shows that topical tacrolimus decreases macrophage infiltrationin post-surgical lymphedema. Representative 40× images of tail tissuesections from control and early topical tacrolimus treated animalsharvested 6 weeks after surgery with immunofluorescent localization ofF4/80⁺ cells are shown in the upper panels. Quantification for bothearly and late treatment experiments is shown below.

FIG. 9 shows the popliteal lymph node dissection model. (A) shows thepopliteal lymph node, filled with Evan's Blue contrast, visible in thepopliteal fat pad. (B) shows that the popliteal lymph node, togetherwith afferent and efferent collectors, is isolated with its surroundingfat pad. (C) shows Evan's blue contrast spilling freely in the surgicalsite, following surgical resection.

FIG. 10 shows that PLND results in increased ROS and DAMPS as comparedto Sham control limbs. (A) shows representative mouse images (leftpanel) showing luminescence (indicating ROS) in hindlimb immediatelyafter PLND (6 hrs) and 1 wk later. Quantification of the luminescentphotons (right panel) shows significantly increased ROS levels in PLNDareas but not in sham at lwk. (B) shows representative immunofluorescentimages of hind limb skin sections from sham and PLND mice stained forHSP-70 (top panel) and HMGB1 (lower panel).

FIG. 11 shows that topical tacrolimus decreases perilymphatic CD4⁺ cellinfiltration after PLND. The upper panels show representative images ofimmunofluorescent localization of CD4⁺ cells and lymphatic vessels(LYVE-1⁺) in animals treated with control or tacrolimus and harvested 4weeks after PLND. Quantification is shown below.

FIG. 12 shows that topical tacrolimus decreases perilymphatic F4/80⁺cell infiltration after PLND. (A) shows representative immunofluorescentimages of tacrolimus and vehicle treated PLND hindlimb skin tissuessections stained for lymphatic vessels (LYVE-1) and macrophages (F4/80).(B) shows quantification of the perilymphatic F4/80+ macrophages.

FIG. 13 shows that topical tacrolimus does not alter collectinglymphatic vessel pulsation or lymphangiogenesis in the absence oflymphatic injury or inflammation. Upper panels are representative imagesof NIR lymphatic images of the hind limb lymphatics in sham operatedmice (i.e., anesthesia without incision or PLND) following treatmentwith control or topical tacrolimus for 2 weeks. Quantification ofcollecting lymphatic vessel pulsation frequency following 3 or 14 daysof treatment with tacrolimus is shown below.

FIG. 14 shows that topical tacrolimus treatment does not alter vascularpermeability following lymphatic injury. (A) shows representative grossimages of tacrolimus or vehicle treated PLND mice hindlimbs after tailvein Evans blue injections to measure vascular permeability. (B) showsquantification of absorbance of formamide extracted Evans blue fromtacrolimus and vehicle treated PLND hind limb tissues.

FIG. 15 shows that topical tacrolimus after PLND does not alter α-SMAcoverage or luminal diameter of hind limb lymphatic collectors. (A)shows (from left to right) brightfield image of the lateral aspect of amouse hind limb with the level of the cross-sections shown by the yellowellipse; NIR image of a mouse hind limb showing the anatomy of lymphaticvessels of the hind limb, with two large-caliber vessels on the lateralaspect; 5× IF image of a mouse hind limb with a yellow box indicatingthe anterolateral leg, where the two dominant collecting lymphaticvessels are located; 20× image of the region in which the dominantcollecting vessels are located (shown with white arrows). (B) showsrepresentative 100× images of cross-sections of the collecting lymphaticvessels after dual immunofluorescent staining for Podoplanin and α-SMA.(C) shows quantification of luminal area. (D) shows quantification ofthe thickness of α-SMA.

FIG. 16 shows that topical tacrolimus does not increaselymphangiogenesis in the absence of injury/inflammation. Upper panelsshow representative 5× images of immunofluorescent staining of lymphaticvessels in unwounded mouse ears after 4 weeks of application of topicaltacrolimus or vehicle control. Quantification of the LYVE-1⁺ stainingarea is shown below.

FIG. 17 shows that inhibition of TGF-β improves lymphatic function anddecreases perilymphatic accumulation of inflammatory cells afterpopliteal lymph node dissection. (A) shows a representative photographof near infrared images of the distal hind limbs of mice treated withisotype control or TGF-β monoclonal antibodies (mAbs) 4 weeks after PLND(upper panel) and pumping frequency in collecting lymphatics (lowerpanel). Note increased pumping frequency of hind limb collectors inTGF-β mAb treated mice. Also notice dermal back flow (white arrow) incontrol but not TGF-β mAb treated mice. (B) shows quantification ofcollecting lymphatic pumping (pulse) frequency in control and TGF-β mABtreated mice. (C) shows quantification of dermal back flow in controland TGF-β mAB treated mice. (D) shows a representative flow cytometryplot from distal hind limb tissues of control and TGF-β mAb treated mice4 weeks after PLND. Note decreased percentage of CD3+CD4+ cells in TGF-βmAb treated mice. (E) shows representative low and high (inset) powerphotomicrographs of perilymphatic (LYVE-1+) inflammatory cells (CD45+;top) and iNOS+ (bottom) cells in control and TGF-β mAb treated mice. (F)shows quantification of flow cytometry for CD4+ cells in hind limbtissues in control and TGF-β mAb treated mice. (G) shows quantificationof quantification of the number of LYVE-1+ vessels in control and TGF-βmAb treated mice. (H) shows quantification of the number ofperilymphatic CD45+ cells in control and TGF-β mAb treated mice. (I)shows quantification of the number of perilymphatic iNOS+ cells incontrol and TGF-β mAb treated mice.

FIG. 18 shows that systemic pirfenidone improves lymphatic function inthe hind limb after surgical lymphatic injury. (A) (upper panels) showsrepresentative NIR images of hind limbs obtained 50 minutes after distalfoot injection of ICG in mice treated with or without pirfenidone 4weeks after PLND. White arrows show dermal backflow of ICG. Insetphotograph is for orientation. Lower panels show graphicalrepresentations of lymphatic vessel pulsations in hind limb collectingvessels of mice treated with or without pirfenidone 4 weeks after PLND.(B) shows quantification of pulsation frequency (pulses/minute) to theright (n=6 animals/group; *p<0.05). (C) shows quantification of dermalbackflow in control and pirfenidone treated mice. (D) showsquantification of LYVE-1⁺ vessels/0.25 mm² area in distal hind limbtissues of control or pirfenidone-treated animals harvested 4 weeksafter PLND (n=6 animals/group; *p<0.001 for LYVE-1). (E) showsrepresentative low and high (inset) power fluorescent co-localizationimages of inflammatory cells (CD45⁺; upper panels), iNOS⁺ cells (lowerpanels) and lymphatic vessels (LYVE-1⁺) in tissues harvested from thedistal hind limbs of animals treated with or without pirfenidone 4 weeksafter PLND, scale bar=100 μm. Higher-power (80×) images are shown in thelower right corner inset of each figure, scale bar=20 μm. Noteperi-lymphatic accumulation of CD45⁺ and iNOS⁺ cells. (F) showsquantification of peril-lymphatic CD45⁺ cells in control and pirfenidonetreated mice. (G) shows quantification of perilymphatic iNOS⁺ cells/hpfin control and pirfenidone-treated mice.

FIG. 19 shows that systemic and topical pirfenidone decrease mouse taillymphedema and inflammation. (A) shows representative photographs ofmouse tails after systemic and topical treatment with pirfenidone orvehicle control. Treatment was started 7 weeks after tail lymphaticinjury. Note marked improvement in pirfenidone treated mice. (B) showsquantification of mouse tail volumes in control, topical pirfenidone,and systemic pirfenidone treated mice. Note significant reductions inpirfenidone treated mice (arrow shows when pirfenidone treatment wasstarted). (C) shows quantification of fibroadipose tissue deposition incontrol and pirfenidone treated mice. (D) shows quantification of type Icollagen deposition in control and pirfenidone treated mice. (E) showsrepresentative cross sectional photomicrographs of mouse tails treatedwith control or pirfenidone (systemic or topical). (F) showsrepresentative high power photomicrographs of tail cross sectionsstained for type I collagen and lymphatic vessels (LYVE-1). Notedecreased type I collagen deposition in pirfenidone treated mice. (G)shows peak nodal uptake of ⁹⁹Tc after distal tail injection of sulfurcolloid conjugated ⁹⁹Tc. Note increased uptake in sacral lymph nodes inpirfenidone treated ice. (H) shows the rate of lymph node uptake of ⁹⁹Tcafter distal tail injection. Note more rapid uptake in pirfenidonetreated mice. (I) shows Representative high power photomicrographs oftail cross sections stained for leukocytes (CD45⁺) and lymphatic vessels(LYVE-1). Note decreased perilymphatic CD45⁺ cell accumulation inpirfenidone treated mice.

FIG. 20 shows that pirfenidone decreases perilymphatic inflammation andTGF-β expression. (A) shows representative photomicrographs of hind limbsections demonstrating perilymphatic accumulation of CD4+ cells incontrol and pirfenidone-treated animals (systemic treatment shown inleft panels; topical treatment shown in right panels). High power imagesare shown in the inset. (B) shows representative photomicrographs ofhind limb sections demonstrating perilymphatic accumulation of TGF-β1+cells in control and pirfenidone-treated animals (systemic treatmentshown in left panels; topical treatment shown in right panels). (C)shows representative photomicrographs of hind limb sectionsdemonstrating perilymphatic accumulation of SMAD3+ cells in control andpirfenidone-treated animals (systemic treatment shown in left panels;topical treatment shown in right panels). (D) shows quantification ofperilymphatic CD4+ cells in control and pirfenidone-treated mice. (E)shows quantification of perilymphatic TGF-β1+ cells in control andpirfenidone-treated mice. (F) shows quantification of perilymphaticSMAD3+ cells in control and pirfenidone-treated mice. (G) shows ahigh-powered photomicrograph of hind limb collecting vessel from controland pirfenidone treated mice stained for podoplanin, α-SMA, and type Icollagen. Note thickening and proliferation of α-SMA+ cells in controlmice. (H) shows quantification of type I collagen deposition aroundcollecting lymphatics of control and pirfenidone treated mice. (I) showsserum TGF-β1 expression in control and systemic pirfenidone treatedmice. (J) shows serum IFN-γ expression in control and systemicpirfenidone treated mice. (K) shows serum VEGF-C expression in controland systemic pirfenidone treated mice.

FIG. 21 shows that loss of T cell but not myeloid cell TGF-β expressionprevents development of lymphedema after lymphatic injury. (A) showsrelative expression of TGF-β1 mRNA in tail tissues harvested fromcontrol, T cell^(cre) and myeloid^(cre) animals 6 weeks following taillymphatic injury. (B) shows representative photomicrographs of control,myeloid-TGF-β1^(cre), and T cell-TGF-β1^(cre) mice. Note lack ofswelling in T cell^(cre) mice. (C) shows quantification of mouse tailvolumes in various groups. Note decreased tail volumes in T cell^(cre)mice as compared to wild-type controls. (D) shows representativephotomicrographs of tail cross sections stained for H&E (top), type Icollagen/LYVE-1 (middle) and quantification of fibroadipose tissuedeposition and Type I collagen expression (lower) in various groups.Note decreased fibroadipose tissue deposition in T cell^(cre) mice. (E)shows peak nodal uptake of ⁹⁹Tc injected in the distal tail. Noteincreased uptake in T cell^(cre) mice. (F) shows rate of sacral nodaluptake of ⁹⁹Tc in various groups. Note more rapid uptake in T cell^(cre)mice.

FIG. 22 shows that mice lacking T cells that express TGF-β1 havedecreased perilymphatic inflammation and TGF-β1 expression. (A) showsrepresentative photomicrographs of mouse hind limbs from various groupsstained for CD4+ cells (top), IL13+ cells (middle), pSMAD3+ cells(bottom), and lymphatic vessels (LYVE-1+). Note decreased CD4+ cellaccumulation, decreased number of IL13+ cells, and decreased number ofpSMAD3+ cells in T Cell mice. (B) shows quantification of CD4⁺ cells inhind limb tissues of animals in various groups. (C) shows quantificationof Th2 cells (CD4+/IL13+) in hind limb tissues of animals in variousgroups. (D) shows quantification of pSMAD3⁺ cells in hind limb tissuesof animals in various groups. (E)-(G) show serum levels of IFN-γ (E),TGF-β1 (F), and VEGF-C (G) protein concentration from control,Myeloid^(Cre), and T Cell^(cre) mice.

FIG. 23 shows that impaired LEC TGF-β1 responsiveness has no change intail lymphedema, inflammation, or fibrosis, but has improvedlymphangiogenesis. (A) shows representative high power (80×) images oflymph node sections harvested from control and FLT4^(cre) mice withimmunofluorescent co-localization of pSMAD⁺ and LYVE-1, scale bar=10 μm.Arrows indicate co-localization of pSMAD3 in LYVE-1⁺ vessels. (B) showsquantification of tail TGF-β1 protein concentration in control andFLT4^(cre) mice 6 weeks after surgery (n=5 animals/group; p=NS forboth). (C) shows representative photographs of control and FLT4^(cre)mouse tails after surgical excision of superficial/deep collectinglymphatics 6 weeks after surgery. (D) shows a graphical representationof tail volume changes of FLT4^(cre) mouse tails as compared withcontrols (n=5 animals/group; p=NS). (E) shows quantification of softtissue changes of control and FLT4^(cre) mice (n=5 animals/group; p=NS).(F) shows quantification of collagen I-staining area (n=5 animals/group;p=NS). (G) (upper panels) shows representative cross-sectionalhistological images of control and FLT4^(cre) mouse tails harvested 6weeks after lymphatic ablation. Brackets illustrate soft tissuethickness. Scale bar=500 μm. (G) (middle panels) shows representative40× images of tail tissues harvested from control and FLT4^(cre) animals6 weeks after surgery with immunofluorescent localization of type Icollagen and lymphatic vessels. (G) (lower panels) shows localization ofpSMAD3 and lymphatic vessels. Scale bar=100 μm. (H) shows representativehigher-power (60×) images of longitudinal tail tissue sections harvestedfrom tail wounds of control and FLT4^(cre) mice 6 weeks after surgerywith immunofluorescent localization of LYVE-1, scale bar=50 μm.Quantifications of bridging LYVE-1⁺ vessels density (LVD) (n/0.25 mm²area) for control and FLT4^(cre) mice (n=5 animals/group; *p<0.01).

FIG. 24 shows that teriflunomide decreases lymphedema. (A) shows a grossphotomicrograph of mouse tails treated with vehicle (control) or topicalteriflunomide 6 weeks following lymphatic ablation. (B) shows a changein tail volume 6 weeks after lymphatic ablation in animals treated withvehicle or topical teriflunomide ((*p<0.0002). (C) shows histologicalcross-sections of mouse tails harvested 1 cm distal to the zone oflymphatic injury in control and teriflunomide treated mice. Bracketsshow area of fibroadipose tissue deposition. (D) shows quantification offibroadipose tissue deposition in tail cross-sections of mice treatedwith vehicle control or topical teriflunomide (*p<0.0001).

FIG. 25 shows that teriflunomide decreased fibrosis. (A) shows arepresentative photomicrograph of mouse tail cross-sections from controland teriflunomide treated mice localizing type I collagen fibers anddermal lymphatics. Notice marked decrease fibrosis in teriflunomidetreated animals. (B) shows quantification of Type I collagen depositionin mice tails treated with vehicle control or topical teriflunomide(*p<0.001). (C) shows a representative photomicrograph of a main hindlimb collecting vessel in animals treated with control or teriflunomidelocalizing α-SMA and podoplanin. Note decreased proliferation of α-SMApositive cells and wider lumen of collecting lymphatics in teriflunomidetreated mice. (D) shows quantification of peri-lymphatic smooth musclethickness in control and teriflunomide treated mice following PLND(*p<0.05).

FIG. 26 shows that teriflunomide decreases inflammation. (A) shows arepresentative photomicrograph of mouse tail cross-sections from controland teriflunomide treated mice, localizing CD4+ cells and dermallymphatics. There is a marked decrease in CD4+ cell infiltration interiflunomide-treated animals. Box insert shows a high power view (80×).(B) shows quantification of CD4+ cells in mice tails treated withvehicle control or topical teriflunomide (*p<0.0001).

FIG. 27 shows that teriflunomide increases lymphangiogenesis. (A) showsa representative photomicrograph of near infra-red imaging of mouse hindlimb lymphatics and collateral vessel formation (white circles) inanimals treated with vehicle control or teriflunomide. Newly formedcollateral lymphatics bypass the popliteal lymph node interiflunomide-treated mice. (B) shows quantification of collaterallymphatic formation in animals following PLND and treated with vehiclecontrol or teriflunomide (*p<0.001). (C) shows a representativephotomicrograph of the tail wound in mice treated with vehicle controlor topical teriflunomide localizing newly formed crossing lymphaticvessels. There is a marked increase in lymphangiogenesis interiflunomide-treated mice. (D) shows quantification of collaterallymphatics in mouse tail wounds of control and teriflunomide treatedanimals 6 weeks after lymphatic ablation (*p<0.001).

FIG. 28 shows that topical teriflunomide decrease lymphatic leakiness.Representative near infra-red image of lymphatic vessels in the hindlimb of mice treated with vehicle control or teriflunomide after PLND.Note decreased leakiness of lymphatic vessels (arrows) interiflunomide-treated mice.

FIG. 29 shows that topical teriflunomide increases lymphatic function.(A) shows a representative flow cytometry plot of trafficked dendriticcells (DCs) in inguinal lymph nodes of mice treated with vehicle controlor teriflunomide following PLND. Mice were treated with a topicalformulation of FITC in the distal hind limb to tag tissue resident DCsand 24 hours later the inguinal lymph nodes were harvested and analyzedusing flow cytometry to quantify the number of DCs that had traffickedfrom the periphery. Marked increase in DC trafficking indicates improvedlymphatic function in teriflunomide-treated animals. (B) showsquantification of DC trafficking in control and teriflunomide-treatedanimals (n=6; *p<0.0001).

FIG. 30 shows that topical teriflunomide increases lymphatic pumping.(A) shows a plot of hind limb collecting lymphatic pumping in controland teriflunomide treated mice following PLND. (B) shows quantificationof hind limb collecting lymphatic pumping frequency in control andteriflunomide-treated mice following PLND. Note significant increase inpumping in teriflunomide treated animals (*p<0.002).

FIG. 31 shows that captopril treatment increases trafficking ofdendritic cells after lymphatic injury. (A) shows representative flowcytometry from inguinal lymph nodes demonstrating FITC+CD11c cells incontrol and captopril treated mice after PLND. (B) shows the percentage(left) and absolute number (right) of FITC+CD11c cells in inguinal lymphnodes of control and captopril treated mice after PLND.

FIG. 32 show that captopril treatment increases hind limb collectinglymphatic pumping. (A) shows a representative plot of collectinglymphatic pumping as assessed by ICG lymphangiography in control andcaptopril treated mice after PLND. (B) shows quantification of packetfrequency (pumping) of hind limb collecting lymphatics.

FIG. 33 shows that captopril treatment decreases T cell infiltrationafter PLND. Representative high power photomicrographs of control (A)and captopril treated (B) mouse hind limb sections stained for CD3+ (Tcell marker) and LYVE-1 (lymphatic marker) are shown. Nuclearcounterstain is shown with DAPI. (C) shows quantification of CD3+ cellsin hind limb tissues of control and captopril treated mice.

FIG. 34 shows that captopril treatment decreases macrophage infiltrationafter PLND. Representative high power photomicrographs of control (A)and captopril treated (B) mouse hind limb sections stained for F4/80+(macrophage marker) and LYVE-1 (lymphatic marker) are shown. Nuclearcounterstain is shown with DAPI. (C) shows quantification of f4/80+cells in hind limb tissues of control and captopril treated mice.

FIG. 35 shows that captopril treatment increases lymphangiogenesis afterPLND. Representative high power photomicrographs of control (A) andcaptopril treated (B) mouse hind limb sections stained for LYVE-1(lymphatic marker) are shown. Nuclear counterstain is shown with DAPI.(C) shows quantification of LYVE-1+ vessels in control and captopriltreated mice (*p<0.05).

FIG. 36 shows that captopril treatment decreases foot swelling afterhind limb lymphatic ablation with DT. Representative photographs ofmouse feet in control (top) and Captopril treated (bottom) groups atvarious times following DT lymphatic ablation. Note obvious decrease infoot swelling in captopril treated mice.

FIG. 37 shows that captopril treatment decreases T cell infiltrationafter hind limb lymphatic ablation with DT. Representative high powerphotomicrographs of control (A) and captopril treated (B) mouse hindlimb sections stained for CD3+ (T cell marker) and LYVE-1 (lymphaticmarker). Ipsilateral tissues are harvested from the limb treated with DTwhile contralateral tissues are from the opposite untreated limb.Nuclear counterstain is shown with DAPI. (C) shows Quantification ofCD3+ cells in hind limb tissues of control and captopril treated mice.Note significant difference (*p<0.05) between ipsilateral control andipsilateral captopril limbs.

FIG. 38 shows that captopril treatment decreases hind limb macrophageinfiltration after lymphatic ablation with DT. Representative high powerphotomicrographs of control (A) and captopril treated (B) mouse hindlimb sections stained for F4/80 (macrophage marker) and LYVE-1(lymphatic marker) are shown. Nuclear counterstain is shown with DAPI.(C) shows quantification of F4/80+ cells in hind limb tissues of controland captopril treated mice. Note significant difference (*p<0.005)between ipsilateral control and ipsilateral captopril limbs.

FIG. 39 shows that captopril treatment decreases hind limb collectingvessel smooth muscle deposition after lymphatic ablation with DT.Representative high power photomicrographs of control (A) and captopriltreated (B) mouse hind limb sections stained for alpha smooth muscleactin (α-SMA) (smooth muscle marker) and podoplanin (lymphatic marker).Nuclear counterstain is shown with DAPI. (C) shows quantification ofα-SMA+ cells in hind limb tissues of control and captopril treated mice.Note significant difference (*p<0.05) between ipsilateral control andipsilateral captopril limbs.

FIG. 40 show that captopril treatment decreases hind limb type Icollagen deposition after lymphatic ablation with DT. Representativehigh power photomicrographs of control (A) and captopril treated (B)mouse hind limb sections stained for type I collagen (fibrosis marker)and LYVE-1 (lymphatic marker) are shows. Nuclear counterstain is shownwith DAPI. (C) shows Quantification of type I collagen in hind limbtissues of control and captopril treated mice. Note significantdifference (*p<0.0001) between ipsilateral control and ipsilateralcaptopril limbs.

FIG. 41 show that captopril treatment decreases hind limb angiotensinconverting enzyme (ACE) expression after lymphatic ablation with DT.Representative high power photomicrographs of control (A) and captopriltreated (B) mouse hind limb sections stained for ACE and LYVE-1(lymphatic marker). Nuclear counterstain is shown with DAPI. (C) showsquantification of ACE in hind limb tissues of control and captopriltreated mice. Note significant difference (*p<0.0001) betweenipsilateral control and ipsilateral captopril limbs.

FIG. 42 shows that captopril treatment increases formation of collaterallymphatics after hind limb lymphatic ablation with DT. RepresentativeICG photographs of control (left panel), Captopril (middle), and normallymphatic architecture (i.e., no DT treatment) hind limbs are shown.Dotted circle represents area in which collateral lymphatics form todrain into inguinal lymph nodes.

FIG. 43 shows that captopril treatment decreases hind limblymphangiogenesis after lymphatic ablation with DT. Representative highpower photomicrograph of control (A) and captopril treated (B) mousehind limb sections stained for LYVE-1 (lymphatic marker). Nuclearcounterstain is shown with DAPI. (C) shows quantification of LYVE-1+lymphatic vessels in hind limb tissues of control and captopril treatedmice. Note significant difference (*p<0.0001) between ipsilateralcontrol and ipsilateral captopril limbs. (D) shows quantification ofLYVE-1+ lymphatic vessel area in hind limb tissues of control andcaptopril treated mice. Note significant decrease (*p<0.005) betweenipsilateral control and ipsilateral captopril limbs.

FIG. 44 shows that captopril treatment decreases limb volumes after hindlimb lymphatic ablation with DT. Quantification of hind limb volumes incontrol and captopril treated mice after lymphatic ablation with DT.Note marked decrease in captopril treated mice (*p<0.007).

FIG. 45 shows that captopril treatment decreases mouse tail lymphedema 6weeks after lymphatic ablation. (A) shows representative photographs ofmouse tails pre-op and weekly, following lymphatic ablation andtreatment with vehicle control or captopril. Arrows indicate timing oftreatment initiation. (B) shows quantification of change in mouse tailvolumes in control and captopril treated mice.

FIG. 46 shows that captopril treatment decreases fibroadipose tissuedeposition in the mouse tail model of lymphedema 6 weeks after lymphaticablation. (A) shows representative H&E stained mouse tail cross sections6 weeks after lymphatic ablation and treated with control (left) orcaptopril topically. Brackets represent fibroadipose tissue deposition.(B) shows Quantification of subcutaneous fibroadipose deposition in micetreated with control or captopril topically 6 weeks after tail lymphaticablation. Note significant decrease in captopril treated mice(*p<0.0001).

FIG. 47 shows that captopril treatment increases mouse tail collectingvessel pumping frequency 6 weeks after lymphatic ablation.Representative photographs of indocyanine green analysis (top) of mousetails treated with control (A) or captopril ointment (B) topically for 6weeks are shown. Line graphs representing collecting lymphaticcontractions are shown below the photographs. Note increased number ofcontractions in captopril treated animals. (C) shows quantification ofcontraction frequency or mouse tail collecting lymphatics in control andcaptopril treated animals. Note increase frequency in captopril treatedmice (*p<0.01).

FIG. 48 shows that captopril treatment increases the number of lymphaticvessels crossing the region of the tail wound in the mouse tail model oflymphedema 6 weeks after lymphatic ablation. Representative longitudinalsections of the mouse tails in control (A) and captopril (B) treatedanimals stained for LYVE-1 (a lymphatic marker) are shown. (C) showsquantification of the LYVE-1+ vessel density in control and captopriltreated mice 6 weeks after tail lymphatic ablation.

FIG. 49 shows that captopril treatment decreases lymphatic vessel areaand correlates with decreased lymphatic stasis in the mouse tail modelof lymphedema 6 weeks after lymphatic ablation. Representative crosssections of the mouse tails in control (A) and captopril (B) treatedanimals stained for LYVE-1 (a lymphatic marker) are shown. Notedecreased diameter of lymphatic vessels in captopril treated mice. (C)shows quantification of LYVE-1+ vessel area in control and captoprilmice. Note significant decrease in lymphatic vessel area correspondingto decreased lymphatic stasis.

FIG. 50 shows that captopril treatment decreases dermal fibrosis andtype I collagen deposition in the mouse tail model of lymphedema 6 weeksafter lymphatic ablation. Representative cross sections of the mousetails in control (A) and captopril (B) treated animals stained for typeI collagen, LYVE-1, and DAPI are shown. Note decreased fibrosis incaptopril treated animals. (C) shows quantification of mouse tail dermaltype I collagen staining area in control and captopril treated mice.Note significant decrease in captopril treated animals (*p<0.0001).

FIG. 51 shows that captopril treatment decreases angiotensin convertingenzyme (ACE) expression in the mouse tail model of lymphedema 6 weeksafter lymphatic ablation. Representative cross sections of the mousetails in control (A) and captopril (B) treated animals stained for ACE,LYVE-1, and DAPI are shown. Note decreased ACE expression in captopriltreated animals. (C) shows quantification of mouse tail ACE stainingarea in control and captopril treated mice. Note significant decrease incaptopril treated animals (*p<0.0001).

FIG. 52 shows that captopril treatment decreases perilymphaticaccumulation of T cells (CD3+) in the mouse tail model of lymphedema 6weeks after lymphatic ablation. Representative cross sections of themouse tails in control (A) and captopril (B) treated animals stained forCD3, LYVE-1, and DAPI are shown. Note decreased ACE expression incaptopril treated animals. (C) shows quantification of mouse tailperilymphatic CD3+ cells/vessel in control and captopril treated mice.Note significant decrease in captopril treated animals (*p<0.0001).

FIG. 53 shows that captopril treatment decreases perilymphaticaccumulation of macrophages (F4/80+ cells) in the mouse tail model oflymphedema 6 weeks after lymphatic ablation. Representative crosssections of the mouse tails in control (A) and captopril (B) treatedanimals stained for F4/80, LYVE-1, and DAPI are shown. Note decreasedACE expression in captopril treated animals. (C) shows quantification ofmouse tail perilymphatic F4/80 cells/vessel in control and captopriltreated mice. Note significant decrease in captopril treated animals(*p<0.0001).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in part, to the use of anti-T cell agentsand/or anti-TGF-β1 agents and/or anti-angiotensin agents as novel, safe,and effective treatments for edema, especially lymphedema. The presentinvention is based, in part, on the surprising discovery that systemicor local administration of anti-T cell, anti-TGF-β1, and/oranti-angiotensin agents, for example, tacrolimus, pirfenidone,teriflunomide, leflunomide, and/or captopril, dramatically improveslymphedema and lymphatic function, and has a variety of other beneficialbiological effects, including stimulating lymphangiogenesis, whenadministered to mammalian subjects. Moreover, because these agents actat different steps of the fibrosis pathway, combinations of anti-T cell,anti-TGF-β1, and/or anti-angiotensin agents can be more effective thanadministration of a single agent, potentially exhibiting synergisticeffects.

Accordingly, the present invention provides compositions and methods fortreating or preventing edema, such as lymphedema, and/or for producing avariety of other beneficial biological effects including, but notlimited to: reduced tissue swelling, reduced lymphatic fluid stasis or“pooling,” reduced tissue fibrosis, reduced tissue inflammation, reducedinfiltration of leukocytes, reduced infiltration of macrophages, reducedinfiltration of naïve and differentiated T-cells, reduced TGF-β1expression and reduced expression and/or activation of downstreammediators (e.g., pSmad3), reduced levels of angiotensins and/or ACE,reduced collagen deposition and/or scar formation, improved or increasedlymphatic function, improved or increased lymph fluid transport,improved or increased lymphangiogenesis, and/or improved or increasedlymph pulsation frequency.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. For example, The Dictionaryof Cell and Molecular Biology (5th ed. J. M. Lackie ed., 2013), theOxford Dictionary of Biochemistry and Molecular Biology (2d ed. R.Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicineand Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skillwith general definitions of some terms used herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, unless the contextclearly dictates otherwise. The terms “a” (or “an”) as well as the terms“one or more” and “at least one” can be used interchangeably.

Furthermore, “and/or” is to be taken as specific disclosure of each ofthe two specified features or components with or without the other.Thus, the term “and/or” as used in a phrase such as “A and/or B” isintended to include A and B, A or B, A (alone), and B (alone). Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to include A, B, and C; A, B, or C; A or B; A or C; B or C; Aand B; A and C; B and C; A (alone); B (alone); and C (alone).

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Where a numeric term is preceded by “about,”the term includes the stated number and values ±10% of the statednumber. The headings provided herein are not limitations of the variousaspects or embodiments of the invention, which can be had by referenceto the specification as a whole. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification in its entirety.

Wherever embodiments are described with the language “comprising,”otherwise analogous embodiments described in terms of “consisting of”and/or “consisting essentially of” are included.

The term “edema,” as used herein, includes lymphedema, lymphaticdysfunction, lymphatic tissue fibrosis, idiopathic edema, peripheraledema, and eye edema. As used herein, “edema” does not include pulmonaryedema or cerebral edema. Edema can include acute edema, chronic edema,post-operative edema, and gradual-onset edema. Symptoms of edema caninclude swelling, fullness, or puffiness of tissues, inflammation,fibrosis, heaviness, pain, decreased range of motion, aching, recurringinfections, skin thickening, or discomfort.

An “active agent” is an agent which itself has biological activity, orwhich is a precursor or prodrug that is converted in the body to anagent having biological activity. Active agents for treating orpreventing edema can include immunosuppressive agents, anti-fibroticagents, anti-T cell agents, anti-TGF-β1 agents, and anti-angiotensinagents. In some embodiments, the agents are small molecule compounds. Inother embodiments, the agents are macromolecules, such aspolynucleotides (e.g., inhibitory RNA) or polypeptides (e.g.,antibodies).

An “anti-T cell agent” is a molecule that reduces T cell-mediatedinflammation, T cell activation, T cell differentiation, and/or T cellproliferation. Classes of anti-T cell agents include calcineurininhibitors and IL-2 inhibitors. Examples of small molecule anti-T cellagents include tacrolimus, teriflunomide, leflunomide, cyclosporine, andpimecrolimus. Examples of macromolecule anti-T cells agents includedenileukin diftitox and Basiliximab.

An “anti-TGF-β1 agent” is a molecule that inhibits the expression,secretion, activation, signaling, or activity of transforming growthfactor beta 1. Pirfenidone is one example of a small moleculeanti-TGF-β1 agent.

An “anti-angiotensin agent” is a molecule that inhibits the activity ofAngI or AngII, or a molecule that inhibits AngI to AngII conversion(e.g., ACE inhibitors). Examples of anti-angiotensin agents includecaptopril, zofenopril, enalapril, lisinopril, ramipril, quinapril,perindopril, benazepril, imidapril, trandolapril, cilazapril,fosinopril, losartan, irbesartan, olmesartan, candesartan, telmisartan,valsartan, fimasartan, diminazene aceturate, xanthenone, and AVE 099.

The terms “inhibit,” “block,” and “suppress” are used interchangeablyand refer to any statistically significant decrease in biologicalactivity, including full blocking of the activity.

In one aspect, the method of the invention can comprise administering acombination of anti-T cell, anti-TGF-β1, and/or anti-angiotensin agents.In a particular embodiment, the method comprises administering apharmaceutical composition of the invention comprising: (i) an effectiveamount of one or more anti-T cell agents selected from the groupconsisting of tacrolimus, teriflunomide, leflunomide, cyclosporine,pimecrolimus, denileukin diftitox, and Basiliximab; and (ii) aneffective amount of one or more anti-TGF-β1 agents and/or one or moreanti-angiotensin agents selected from the group consisting ofpirfenidone, captopril, zofenopril, enalapril, lisinopril, ramipril,quinapril, perindopril, benazepril, imidapril, trandolapril, cilazapril,and fosinopril, losartan, irbesartan, olmesartan, candesartan,telmisartan, valsartan, and fimasartan. The method of the invention cancomprise administering a pharmaceutical compound of the inventioncomprising any combination of anti-T cell, anti-TGF-β1, and/oranti-angiotensin agents. For instance, in one embodiment, the methodcomprises administering a pharmaceutical composition comprisingtacrolimus and pirfenidone. In another embodiment, the method comprisesadministering a pharmaceutical composition comprising tacrolimus,pirfenidone, and teriflunomide. In an additional embodiment, the methodcomprises administering a pharmaceutical composition comprisingtacrolimus, pirfenidone, and leflunomide. In further aspects, the methodcomprises administering a pharmaceutical composition comprisingtacrolimus and captopril; or teriflunomide and captopril; or leflunomideand captopril; or pirfenidone and captopril; or tacrolimus, captopril,and teriflunomide; or tacrolimus, captopril, and leflunomide; ortacrolimus, captopril, and pirfenidone.

By “subject” or “individual” or “patient” is meant any subject,preferably a mammalian subject, for whom diagnosis, prognosis, ortherapy is desired. Mammalian subjects include humans, domestic animals,farm animals, sports animals, and zoo animals including, e.g., humans,non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice,horses, cattle, and so on.

In some embodiments the subject may have or may have had cancer, forexample, a cancer comprising a solid tumor. In some embodiments thesubject may have or may have had breast cancer or a cancer affectingfemale reproductive organs, cutaneous system, musculoskeletal system,soft tissues of the extremities or trunk, male reproductive system,urinary system, or the head and neck. In some embodiments, the subjectmay have undergone axillary lymph node dissection. In some embodiments,the subject has received treatment for cancer, and the edema,lymphedema, or lymphatic injury is associated with the cancer treatmentor diagnosis. For example, the subject may be receiving or may havereceived chemotherapy or radiation therapy for cancer treatment or otherindications, or may have had one or more lymph nodes surgically removedin the course of cancer treatment or diagnosis.

In some embodiments the subject may have sustained a lymphatic injury(for example as the result of removal, ligation or obstruction of lymphnodes or lymph vessels, or fibrosis of lymph tissue), or the subject maybe obese or have or had an infection that leads to edema, such aslymphedema. In some embodiments the infection may be a skin infection ora history of skin infection(s) that are related to lymphedema orlymphatic injury. In some embodiments the infection may be a parasiticinfection that obstructs lymphatic flow or injures the lymphatic system.In some embodiments the subject may have sustained lymphatic injury fromjoint replacement, trauma, burns, radiation, or chemotherapy.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of a diagnosed pathologiccondition or disorder. Thus, those in need of treatment include thosealready with the disorder. In certain embodiments, a subject issuccessfully “treated” for a disease or disorder according to themethods provided herein if the patient shows, e.g., total, partial, ortransient alleviation or elimination of symptoms associated with thedisease or disorder. For example, “treating edema” can include, but isnot limited to, decreasing swelling, decreasing inflammation, decreasingfibrosis, decreasing pain, increasing range of motion, decreasingheaviness, decreasing tightness, decreasing skin thickening, and/orimproving lymphatic function.

“Prevent” or “prevention” refers to prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of preventioninclude those at risk of or susceptible to developing the disorder.Subjects that are at risk of or susceptible to developing lymphedemainclude, but are not limited to, cancer patients undergoing radiationtherapy, chemotherapy, and/or surgical lymph node dissection. In certainembodiments, a disease or disorder is successfully prevented accordingto the methods provided herein if the patient develops, transiently orpermanently, e.g., fewer or less severe symptoms associated with thedisease or disorder, or a later onset of symptoms associated with thedisease or disorder, than a patient who has not been subject to themethods of the invention.

In a prophylactic context, the pharmaceutical composition of theinvention can be administered at any time before or after an event, forexample, radiation therapy, chemotherapy, or surgical lymph nodedissection, which places a subject at risk of or susceptible tolymphatic injury and/or developing edema. In some aspects, thepharmaceutical composition is administered prophylactically up to aboutone week before the event, such as 1, 2, 3, 4, 5, 6, or 7 days beforethe event. In some instances, the pharmaceutical composition isadministered prophylactically on the same day as the event. In someembodiments, the pharmaceutical composition is administeredprophylactically within six weeks of the event, for example, withinabout 1, 2, 3, 4, 5, or 6 days, or within about 1, 2, 3, 4, 5 or 6 weeksof the event. In one embodiment, the pharmaceutical composition isadministered prophylactically for about 2-4 weeks or for about 1, 2, 3,4, 5, or 6 weeks.

In some embodiments the treatment and/or prevention methods describedherein may be performed in combination with one or more additional edemaor lymphedema treatment and/or prevention methods known in the art, forexample, treatment methods involving the administration of othertherapeutic agents and/or treatment methods involving surgery, massage,compression therapy, fluid drainage therapy, acupuncture, laser, or anyother suitable treatment methods.

The term “pharmaceutical composition” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective, and which contains no additional components that areunacceptably toxic to a subject to which the composition would beadministered. Pharmaceutical compositions can be in numerous dosageforms, for example, tablet, capsule, liquid, solution, softgel,suspension, emulsion, syrup, elixir, tincture, film, powder, hydrogel,ointment, paste, cream, lotion, gel, mousse, foam, lacquer, spray,aerosol, inhaler, nebulizer, ophthalmic drops, patch, suppository,and/or enema. Pharmaceutical compositions typically comprise apharmaceutically acceptable carrier, and can comprise one or more of abuffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), a stabilizing agent (e.g. human albumin), a preservative(e.g. benzyl alcohol), a penetration enhancer, an absorption promoter toenhance bioavailability and/or other conventional solubilizing ordispersing agents. Choice of dosage form and excipients depends upon theactive agent to be delivered and the disease or disorder to be treatedor prevented, and is routine to one of ordinary skill in the art.

“Systemic administration” means that a pharmaceutical composition isadministered such that the active agent enters the circulatory system,for example, via enteral, parenteral, inhalational, or transdermalroutes. Enteral routes of administration involve the gastrointestinaltract and include, without limitation, oral, sublingual, buccal, andrectal delivery. Parenteral routes of administration involve routesother than the gastrointestinal tract and include, without limitation,intravenous, intramuscular, intraperitoneal, intrathecal, andsubcutaneous. “Local administration” means that a pharmaceuticalcomposition is administered directly to where its action is desired(e.g., at or near the site of the injury or symptoms). Local routes ofadministration include, without limitation, topical, inhalational,subcutaneous, ophthalmic, and otic. It is within the purview of one ofordinary skill in the art to formulate pharmaceutical compositions thatare suitable for their intended route of administration.

An “effective amount” of a composition as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose, route of administration, and dosageform.

In some embodiments, administration of the anti-T cell agent and/or theanti-TGF-β1 agent and/or the anti-angiotensin agent can comprisesystemic administration, at any suitable dose and/or according to anysuitable dosing regimen, as determined by one of skill in the art. Forexample, in some embodiments, tacrolimus or an analogue, variant, orderivative thereof is administered systemically to the subject at adaily dose of about 0.01 mg/kg to about 5 mg/kg. More particularly,tacrolimus or an analogue, variant, or derivative thereof can beadministered to the subject at a daily dose of about 0.01, 0.015, 0.02,0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075,0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75,3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mg/kg.

In some embodiments, teriflunomide or an analogue, variant, orderivative thereof is administered systemically to the subject at adaily dose of about 0.1 mg/kg to about 5 mg/kg. More particularly,teriflunomide or an analogue, variant, or derivative thereof can beadministered to the subject at a daily dose of about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75,or 5.0 mg/kg.

In some embodiments, leflunomide or an analogue, variant, or derivativethereof is administered systemically to the subject at a daily dose ofabout 0.1 mg/kg. to about 5 mg/kg. More particularly, leflunomide or ananalogue, variant, or derivative thereof can be administered to thesubject at a daily dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5,2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mg/kg.

In some embodiments, pirfenidone or an analogue, variant, or derivativethereof is administered systemically to the subject at a daily dose ofabout 50 mg/kg to about 2500 mg/kg. More particularly, pirfenidone or ananalogue, variant, or derivative thereof can be administered to thesubject at a daily dose of about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260,280, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, or 2500 mg/kg.

In some embodiments, captopril or an analogue, variant, or derivativethereof is administered systemically to the subject at a daily dose ofabout 0.1 mg/kg to about 10 mg/kg. More particularly, captopril or ananalogue, variant, or derivative thereof can be administered to thesubject at a daily dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5,2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg/kg.

In some embodiments, administration of the anti-T cell agent and/or theanti-TGF-β1 agent and/or the anti-angiotensin agent can comprise localadministration, at any suitable dose and/or according to any suitabledosing regimen, as determined by one of skill in the art. For example,in some embodiments, tacrolimus or an analogue, variant, or derivativethereof is administered to the subject in the form of a topicalcomposition comprising from about 0.01 mg/ml to about 5 mg/ml, or fromabout 0.1 mg/ml to 2 mg/ml, or about 0.01, 0.015, 0.02, 0.025, 0.03,0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085,0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5,3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mg/ml tacrolimus or an analogue,variant, or derivative thereof. In some embodiments, tacrolimus or ananalogue, variant, or derivative thereof is administered to the subjectin the form of a topical composition comprising from about 0.01% toabout 1%, or from about 0.03% to about 0.5%, or from about 0.05 to about0.2%, or about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15,0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8,0.85, 0.9, 0.95, or 1.0% tacrolimus or an analogue, variant, orderivative thereof.

In some embodiments, teriflunomide or an analogue, variant, orderivative thereof is administered to the subject the form of a topicalcomposition comprising from about 10 mg/ml to about 50 mg/ml, or about20 mg/ml to about 30 mg/ml, or about 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg/mlteriflunomide or an analogue, variant, or derivative thereof. In someembodiments, teriflunomide or an analogue, variant, or derivativethereof is administered to the subject the form of a topical compositioncomprising from about 1% to about 20%, or from about 5% to about 15%, orabout 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5,2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 15, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20%teriflunomide or an analogue, variant, or derivative thereof.

In some embodiments, leflunomide or an analogue, variant, or derivativethereof is administered to the subject the form of a topical compositioncomprising from about 10 mg/ml to about 50 mg/ml, or about 20 mg/ml toabout 30 mg/ml, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg/ml leflunomide or ananalogue, variant, or derivative thereof. In some embodiments,leflunomide or an analogue, variant, or derivative thereof isadministered to the subject the form of a topical composition comprisingfrom about 1% to about 20%, or from about 5% to about 15%, or about 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0,3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 15, 14, 14.5, 15,15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20% leflunomide or ananalogue, variant, or derivative thereof.

In some embodiments, pirfenidone or an analogue, variant, or derivativethereof is administered to the subject the form of a topical compositioncomprising from about 0.1 mg/ml to about 5 mg/ml, or from about 0.5mg/ml to 2 mg/ml, or about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75,4.0, 4.25, 4.5, 4.75, or 5.0 mg/ml pirfenidone or an analogue, variant,or derivative thereof. In some embodiments, pirfenidone or an analogue,variant, or derivative thereof is administered to the subject the formof a topical composition comprising from about 0.1% to about 20%, orfrom about 1% to about 10%, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25,2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 15,14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20%pirfenidone or analogue, variant, or derivative thereof.

In some embodiments, captopril or an analogue, variant, or derivativethereof is administered to the subject the form of a topical compositioncomprising from about 0.1 mg/ml to about 5 mg/ml, or from about 0.5mg/ml to about 2 mg/ml, or about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035,0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09,0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5,3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mg/ml captopril or an analogue,variant, or derivative thereof. In some embodiments, captopril or ananalogue, variant, or derivative thereof is administered to the subjectthe form of a topical composition comprising from about 1% to about 20%,or from about 5% to about 15%, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0,4.25, 4.5, 4.75, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10,10.5, 11, 11.5, 12, 12.5, 13, 15, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, or 20% captopril or an analogue, variant, orderivative thereof.

The anti-T cell agent and/or anti-TGF-β1 agent and/or anti-angiotensinagent can be administered according to any suitable dosing regimen, forexample, where the daily dose is divided into two or more separatedoses. It is within the skill of the ordinary artisan to determine adosing schedule and duration for systemic or local administration. Insome embodiments, the pharmaceutical composition is administered orallyat least once a day or at least twice a day. In some embodiments, thepharmaceutical composition is administered intravenously at least once aday or at least twice a day. In some embodiments, the pharmaceuticalcomposition is administered topically at least once a day or at leasttwice a day. In some embodiments, the pharmaceutical composition isadministered subcutaneously at least once a day or at least twice a day.

In embodiments in which more than one active agent is administered, theagents can be administered together (for example, in the sameformulation and/or at the same time), or separately (for example, indifferent formulations and/or at different times). In some suchembodiments, the agents are administered systemically. In some suchembodiments, the agents are administered locally. In some suchembodiments, one (or more) agent is administered systemically and one(or more) agent is administered locally, for example, topically. Wheretwo such agents are used, it is possible to use lower dosages or amountsof each agent, as compared to the dosages necessary when each agent isused alone.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, can be practiced without departing from the scope of thepresent disclosure.

EXAMPLES Example 1 Treatment and Prevention of Lymphedema UsingTacrolimus

Tacrolimus Decreases Tail Lymphedema without Systemic Immunosuppression

To study the effect of topical tacrolimus on lymphedema, we used apreviously described mouse tail model of lymphedema. Goldman et al.,Circ. Res. 96:1193-1199 (2005); Shimizu et al., J. Am. Heart Assoc.2:e000438 (2013); Choi et al., Circulation 125:872-882 (2012);Tabibiazar et al., PLoS Med. 3:e254 (2006); Yoon et al., J. Clin.Invest. 111:717-725 (2003). Disruption of the superficial and deeplymphatics of the mouse tail resulted in a greater than 100% increase intail volumes 2 weeks after surgery (FIG. 1(A), (B)). We have previouslyshown that swelling at this time point is due primarily to accumulationof interstitial fluid. Avraham et al., FASEB J. 27:1114-1126 (2013).Chronic lymphatic obstruction in the tail results in gradual replacementof interstitial fluid by fibroadipose tissue, as well as accumulation ofinflammatory cells occurs over the ensuing 4 weeks. Avraham et al.,FASEB J. 27:1114-1126 (2013). These pathologic changes closely mirrorclinical lymphedema and persist for at least 6-9 additional weeks oncelymphedema is established. Goldman et al., Circ. Res. 96:1193-1199(2005); Shimizu et al., J. Am. Heart Assoc. 2:e000438 (2013); Choi etal., Circulation 125:872-882 (2012); Tabibiazar et al., PLoS Med. 3:e254(2006); Yoon et al., J. Clin. Invest. 111:717-725 (2003).

Based on this knowledge, we used two different tacrolimus treatmentapproaches.

One group of animals were treated with tacrolimus beginning 2 weeksafter surgery for 4 weeks (total of 6 weeks after tail surgery), in aneffort to prevent development of lymphedema (i.e., early treatment). Inanother group we waited 6 weeks after lymphatic ablation for lymphedemato become established and then treated with tacrolimus until 9 weeks,with the intent to treat established soft tissue changes (i.e., latetreatment). In all studies, we treated animals twice daily with either0.1% tacrolimus (0.05 g/application) or vehicle control (petroleumjelly). Tacrolimus or vehicle control were applied as a thin layer tothe entire tail distal to (i.e., not including) the surgical site.

Early treatment with topical tacrolimus markedly decreased tail swellingand prevented development of permanent swelling (FIG. 1(A), B)). Grossexamination of the tails from experimental animals demonstrated a nearcomplete resolution of lymphedema and this change corresponded to a 95%decrease in tail volume and nearly 50% decrease in soft tissue thickness(FIG. 1(B), (C)). Late treatment was also highly effective in decreasinggross tail swelling, tail volume, and soft tissue thickness, as comparedwith controls, although in these animals the tail volumes did not returnto preoperative levels (FIG. 1(B), (C)).

We also analyzed systemic levels of tacrolimus and peripheral T cellcounts to determine if topically applied tacrolimus is absorbed in anappreciable manner. This analysis demonstrated that systemic absorptionof topical tacrolimus (mean value of 1.06 ng mL⁻¹) remainedsignificantly below the known therapeutic immunosuppressive levelsachieved with systemic administration (5-15 ng mL⁻¹; FIG. 1(D)). Inaddition, animals treated with topical tacrolimus showed no changes incirculating blood T cells or CD4⁺ cells (FIG. 1(E), FIG. 7), as comparedto vehicle treated controls.

Tacrolimus Decreases Inflammation and Fibrosis After Lymphatic Injury

Chronic inflammation is a histological hallmark of clinical lymphedemaand is characterized by increased accumulation of T-helper cells, Tregulatory cells, and macrophages. Avraham et al., FASEB J. 27:1114-1126(2013); Zampell et al., PLoS ONE 7:e49940 (2012); Ghanta et al., Am. J.Physiol. Heart Circ. Physiol. 308:H1065-1077 (2015); Olszewski et al.,Lymphology 23:23-33 (1990). Consistent with this, we found thattacrolimus-treated animals had markedly decreased numbers of leukocytesinfiltrating the dermis and subcutaneous fat as compared with controls(CD45⁻ cells; 56% reduction-early treatment; 49% late treatment; FIG. 2(A_(i)), (A_(ii))). Inflammatory cells in lymphedematous tissuesharvested from control animals were located in close proximity to thecapillary and collecting lymphatics, but were virtually absent intacrolimus treated mice. Similarly, we noted marked decreases in thenumbers of infiltrating CD3⁺ cells (53% reduction-early; 49% latetreatment; FIG. 2 (B_(i)), (B_(ii)), CD4⁺ cells (78% reduction-earlytreatment, 71% late treatment; FIG. 2 (C_(i), (C_(ii))), andIFN-γ-producing cells (54% reduction-early treatment, 57% latetreatment; FIG. 2 (D_(i)), (D_(ii))). Additionally, we noted a decreasein the soft tissue infiltration of macrophages (F4/80⁺ cells; 86%reduction-early treatment; 73% late treatment; FIG. 8). Taken together,these findings show that following lymphatic injury, inflammatory cellsaccumulate in large numbers in close proximity to skin/subcutaneouslymphatic vessels and that this response is mitigated by topicalapplication of tacrolimus.

Patients with lymphedema have progressive soft tissue fibrosis and thedegree of fibrosis correlates with the severity of disease. Tassenoy etal., Lymphat. Res. Biol. 7:145-151 (2009). Therefore, we analyzedseveral markers of fibrosis in the tail tissues to understand theeffects of tacrolimus treatment on this aspect of the disease. We foundthat topical treatment with tacrolimus markedly decreased dermal andsubcutaneous type I collagen deposition and Scar index (picrosirius redbirefringence measuring the ratio of collagen (I/III), as compared withcontrol mice (FIG. 3 (A_(i)), (A_(ii)), (B_(i)), (B_(ii)). Lymphaticvessels of control mice were surrounded by thick layers of type Icollagen; in contrast, tacrolimus-treated animals had essentially normallymphatic vessels. Consistent with this observation and our previousreports (Clavin et al., Am. J. Physiol. Heart Circ. Physiol.295:H2113-2127 (2008)), we also found that tacrolimus treatment markedlydecreased expression of the pro-fibrotic growth factor TGF-β1 andcellular expression of its activated downstream signaling molecule,phosphorylated SMAD3 (pSMAD-3; FIG. 3 (C_(i)), C_(ii)), (D_(i)),(D_(ii))). The degree of this response was similar for both early andlate tacrolimus treatments.

Tacrolimus Increases Lymphatic Function

To assess lymphatic function we performed Near infrared (NIR)lymphangiography with indocyanine green (ICG), which has been describedas an effective means of quantifying lymphatic function in humans, pigs,and mice by enabling real-time imaging of lymphatic vessels, calculationof packet frequency (or pulsatile flow of lymphatic fluid), and analysisof dermal back flow and dye clearance. Kwon et al., Lymphat. Res. Biol.5:219-231 (2007); Sharma et al., Am. J. Physiol. Heart Circ. Physiol.292:H3109-3118 (2007); Unno et al., J. Vasc. Surg. 52:946-952 (2010).Using NIR imaging 6 weeks after lymphatic ligation, we noted rapidtransport of interstitial fluid proximally across the tail wound inanimals in which treatment was started 2 weeks after lymphatic injury(early treatment; FIG. 4(A)). In contrast, control animals demonstratedpooling of ICG distal to the lymphatic excision site with no transportacross the zone of injury. This finding was confirmed usingtechnetium-99m (^(99m)Tc) lymphoscintigraphy, a technique in which aradiotracer is injected in the distal tail and uptake by the sacrallymph nodes is measured over time. Decay adjusted uptake of the sacrallymph nodes in the early treatment animals demonstrated a more than6-fold increase in ^(99m)Tc uptake in tacrolimus-treated animals ascompared with controls (FIG. 4 (B_(i)), (B_(ii))). Late treatment withtacrolimus similarly increased nodal uptake (2-fold); however, thisdifference did not reach statistical significance.

Given the efficacy of tacrolimus in preventing and treating lymphedemain the tail model, we next sought to study how tacrolimus modulateslymphatic function after lymphatic injury using a previously describedmodel of popliteal lymph node dissection (PLND) (FIG. 9(A)-(C)). Blum etal., Breast Cancer Res. Treat. 139:81-86 (2013). This model isclinically relevant since lymph node dissection in the course of cancertreatment is the most common cause of lymphedema in developed countries.We first utilized the PLND mouse model to better understand themechanisms by which chronic inflammatory reactions are activated afterlymphatic injury.

Previous studies have demonstrated that lymphatic endothelial cells(LECs) are highly sensitive to reactive oxygen species (ROS) (Kasuya etal., Sci. Rep. 4:4173 (2014)) and that ROS can activate chronicinflammation. Gorlach et al., Redox Biol. 6:372-385 (2015). To determinewhether ROS are present following PLND, we analyzed accumulation of ROSin tissues distal to the zone of injury 1 week after injury based on theknown role of lymphatic vessels in removing cellular metabolic products.Indeed, this analysis demonstrated a significant accumulation of ROS inthe hind limb tissues immediately distal to the popliteal region inanimals treated with PLND (FIG. 10(A)). In contrast, control animalsthat had been treated with skin incision without lymphadenectomy hadvirtually no accumulation of ROS. ROS can activate innate immuneresponses including danger-associated molecular pattern molecules(DAMPs). Yin et al., J. Immunol. 194:429-437 (2015). Consistent withthese studies and our finding of increased ROS after PLND, as well aswith our previous studies using a tail model of lymphedema in which wedemonstrated increased expression of HMGB1 in a variety of cell types inlymphomatous tissues (Zampell et al., Am. J. Physiol. Cell Physiol.300:C1107-1121 (2011)), we noted a marked increase in the expression ofDAMPs such as heat shock protein-70 (HSP70) and high-mobility group box1 (HMGB-1) (FIG. 10(B)). These findings indicate that lymphatic injuryresults in generation of ROS, which in turn, result in cellular injury,expression of DAMPs, and initiation of inflammatory responses.

Previous clinical and laboratory studies have described dermal backflowas pooling of ICG into the interstitial space resulting from leaky,dysfunctional lymphatics. Blum et al., Breast Cancer Res. Treat.139:81-86 (2013); Tashiro et al., Ann. Plast. Surg. doi: 10.1097/SAP.599(2015); Yamamoto et al., Plast. Reconstr. Surg. 128:314e-321e (2011).Consistent with these reports, we found that control animals treatedtopically with petroleum jelly alone for 4 weeks had marked leakiness ofthe initial lymphatics of the foot pad (punctate areas of bright ICGaccumulation indicated by white arrows; FIG. 4(C)) and dermal back flow(generalized retention of ICG in the dermis; FIG. 4(C)). This pathologicresponse was markedly decreased in animals treated with topicaltacrolimus resulting in decreased lymphatic leakiness and improvedclearance of injected ICG.

Analysis of fluctuations in ICG florescence intensity in the collectingvessels using time lapse photography is a technique that has beenpreviously used to measure the rate of lymphatic pumping. Sevick-Muracaet al., J. Clin. Invest. 124:905-914 (2014). This analysis enablescalculation of “ICG packet frequency” and has been used to analyzecollecting lymphatic function after PLND. Blum et al., Breast CancerRes. Treat. 139:81-86 (2013). Using this technique, we found thattopical tacrolimus therapy markedly increased collecting lymphaticpacket frequency as compared with controls (>2-fold increase) indicatingthat this treatment increases collecting lymphatic function (FIG. 4(Di), (Dii)).

In addition, similar to our findings with the tail model, we found thattreatment with topical tacrolimus after PLND markedly decreasedperilymphatic infiltration of inflammatory cells (39% reduction in CD45⁺cells (FIG. 4(E) upper panel, FIG. 4(F)) 56% reduction in CD4⁺ cells(FIG. 11) and 36% reduction in F4/80⁺ cells (FIG. 12(A), (B)) comparedto vehicle treated controls. Treatment with topical tacrolimus alsoresulted in a significant decrease in perilymphatic expression ofinducible nitric oxide synthase (iNOS) by inflammatory cells (42%reduction in the number of iNOS-expressing cells) (FIG. 4 (E) lowerpanel, FIG. 4 (G)). This is important since previous studies have shownthat perilymphatic iNOS expression is an important regulator ofcollecting lymphatic pumping capacity. Liao et al., Proc. Natl. Acad.Sci. USA 108:18784-18789 (2011). Importantly, changes in lymphaticcontractility in response to topical tacrolimus treatment were onlyobserved in the setting of lymphatic injury since treatment ofnon-operated animals (i.e., anesthesia only but no surgery) withtacrolimus did not increase lymphatic contraction frequency orperilymphatic accumulation of inflammatory cells (FIG. 13).

Additionally, to ensure that the observed effects of tacrolimus onlymphatic function were not a result of decreased vascular permeabilityand blood vessel leakage, we performed a Miles assay to measure bloodvessel permeability following PLND surgeries with and withouttacrolimus. We observed no differences in blood vessel permeabilitybetween tacrolimus treated and vehicle control, suggesting that theeffects of tacrolimus in increasing lymphatic function are indeed due toincreased lymphatic function rather than decreased vascular leakage(FIG. 14(A), (B)).

We next examined luminal diameter and alpha smooth muscle cell coverageof the hind limb collecting lymphatics following PLND in control andtacrolimus treated animals based on the fact that previous clinicalreports have shown that patients with long-standing severe lymphedemaconstricted lymphatics with smooth muscle cell hypertrophy. Mihara etal., PLoS One 7:e41126 (2012). Not surprisingly, in this relativelyearly time period after lymphatic injury (i.e., 4 weeks), we found nodifferences in the number of alpha smooth muscles surrounding thevessels or in the luminal area of hind limb collectors when comparingtacrolimus and control treated mice. This finding indicates thatincreases in collecting lymphatic packet frequency after tacrolimustreatment are not regulated by lymphatic structural changes but ratherdue to changes in the lymphatic microenvironment (e.g., perilymphaticinflammation or expression of iNOS; FIG. 15(A)-(D)).

Tacrolimus Increases Collateral Lymphatic Vessel Formation

Because T cells are known to potently inhibit lymph nodelymphangiogenesis (Kataru et al., Immunity 34:96-107 (2011)) andinflammatory lymphangiogenesis during wound repair (Zampell et al., Am.J. Physiol. Cell Physiol. 302:C392-C404 (2012)), we next sought todetermine if treatment with tacrolimus increases the formation ofcollateral lymphatics. Indeed, histological analysis of tail wounds andidentification of lymphatics using LYVE-1 immunofluorescent (IF)staining demonstrated a marked increase in newly formed lymphaticvessels bridging the zone of lymphatic injury (189% increase after earlytreatment; 106% increase after late treatment; FIG. 5 (A_(i)),(A_(ii))). This lymphangiogenic response appeared to be independent ofVEGF-C expression since we noted no differences in VEGF-C mRNAexpression between control and tacrolimus treated animals (FIG. 5(A_(iii))). However, consistent with our IF staining analysis, we noteda significant decrease in the expression of two potentlyanti-lymphangiogenic growth factors and cytokines, TGF-β1 (Oka et al.,Blood 111:4571-4579 (2008); Clavin et al., Am. J. Physiol. Heart Circ.Physiol. 295:H2113-2127 (2008)) and IFN-γ (Kataru et al., Immunity34:96-107 (2011); Shao et al., J. Interferon Cytokine Res. 26:568-574(2006)) (FIG. 5 (A_(iii))). Lymphangiographic analysis by NIR imaging,and IF staining for lymphatic vessels in hind limbs 4 weeks followingPLND, confirmed our tail model findings demonstrating that animalstreated with tacrolimus consistently had significantly more collaterallymphatics draining towards the inguinal lymph node, thereby bypassingthe zone of injury (FIG. 5(B)-(D)).

To determine the lymphangiogenic effects of tacrolimus in otherinflammatory and wound models in which drainage of lymphatic fluid isnot obstructed surgically, we next used two other models of inflammatorylymphangiogenesis. The cornea is a useful tissue for studyinglymphangiogenesis because it is normally devoid of both blood andlymphatic vessels but develops both in the setting of inflammation.Cursiefen et al., J. Clin. Invest. 113:1040-1050 (2004). We placedsutures in the corneas of mice and treated them with systemic tacrolimusor vehicle control daily for 2 weeks and found that tacrolimus treatmentresulted in a significant increase in the proliferation of lymphatic(48% increase) but not blood vessels (FIG. 6(A)-(C)).

We also studied lymphangiogenesis during wound healing using an earpunch wound model and applying topical tacrolimus or control ointmentfor 4 weeks. Similar to the corneal model, we found that topicaltacrolimus significantly increased lymphatic vessel density andbranching in the ear skin adjacent to the wound as compared withcontrols (FIG. 6(D), (F)). To test the possibility of directlymphangiogenic effects of tacrolimus, we applied tacrolimus tounwounded mouse ears for 4 weeks, and then performed whole mountconfocal imaging of lymphatic vessels. We observed no increase inlymphangiogenesis in this uninjured, non-inflamed setting (FIG. 16).Together, these results indicate that tacrolimus facilitates theformation of new lymphatic vessels in the setting of inflammationgenerally and in the setting of lymphedema/lymphatic injuryspecifically.

Methods

Study Design

The aim of this study was to test the hypothesis that local inhibitionof T cells can both prevent development of lymphedema after lymphaticinjury and treat established lymphedema after it has developed. Usingtwo different mouse models of lymphatic injury and lymphedema weanalyzed the efficacy of Tacrolimus, an FDA approved topical anti-T cellmedication on these outcomes. Having found that Tacrolimus was indeedeffective in preventing and treating lymphedema, we then sought toanalyze the cellular and molecular mechanisms that regulate thisresponse. In these subsequent studies we tested the hypothesis thatinhibition of CD4+ inflammatory responses improved lymphatic function byincreasing the formation of collateral lymphatics, decreasing collagendeposition in the extracellular matrix surrounding initial lymphatics,and increasing the pumping frequency of collecting lymphatics. Ourstudies were all performed using adult female (10-14 week old) C57BL/6Jmice (Jackson Laboratories, Bar Harbor, Me.) that were maintained inlight- and temperature-controlled pathogen free environments and fed adlibitum. All studies were approved by the Institutional Animal Care andUse Committee (IACUC) at Memorial Sloan Kettering Cancer Center. Eachexperiment was performed using a minimum of 6-8 animals and assays wereperformed in triplicate. All cell counts were performed by reviewersblinded to the intervention.

Animal Models and Treatments

Tail surgery and lymphatic ablation was performed as previouslypublished. Clavin et al., Am. J. Physiol. Heart Circ. Physiol.295:H2113-H2127 (2008). Briefly, the superficial and deep collectinglymphatics of the mid portion of the tail were excised using a 2 mmcircumferential excision. Tacrolimus 0.1% (Astellas, Tokyo,Japan)/vehicle control (petroleum jelly) was treated topically beginningat 2 weeks after surgery (early treatment) and beginning at 6 weeksafter surgery (late treatment) in different set of animals. For bothapproaches we treated the animals with 0.1% tacrolimus or vehiclecontrol, twice daily for 4 week period (for early treatment) and 3 weekperiod (late treatment). Tacrolimus (approx. 0.05 g) was applied as athin layer to the entire tail area. To enable analysis of the lymphaticcollecting vessel pumping capacity we utilized a previously describedmouse popliteal lymphadenectomy model. Blum et al., Breast Cancer ResTreat 139:81-86 (2013); Sharma et al., Am. J. Physiol. Heart Circ.Physiol. 292:H3109-H3118 (2007). Briefly, the hind limb collectingvessels and popliteal lymph nodes were identified and the lymph nodeswere excised with the popliteal fat pad. Beginning 2 weeks aftersurgery, animals were randomized to treatment with either 0.1% topicaltacrolimus or vehicle control (petroleum jelly) twice daily for 2 weeks.

A corneal lymphangiogenesis assay was performed as previously reported.Cursiefen et al., Cornea 25:443-447 (2006). Briefly, 10-0 nylon sutures(Ethicon, Cincinnati, Ohio) were placed in the cornea at 120° angles.Beginning immediately after suture placement, animals were treated withsystemic tacrolimus or vehicle control daily for two weeks, followed byanalysis using confocal microscopy (Leica Microsystems, Weitziar,Germany). Systemic tacrolimus (Biotang Inc., Lexington, Mass.) wasdissolved in 10% ethanol with 1% tween 80 in PBS(Rozkalne et al.,Neurobiol. Dis. 41:650-654 (2011); Butcher et al., J. Neurosci.17:6939-6946 (1997)) and dosed at 4 mg kg⁻¹ IP daily. Vehicle controlfor systemic tacrolimus was the equivalent volume of 10% ethanol, 1%tween 80 in PBS. Cutaneous lymphangiogenesis was assessed using an earpunch wound model as previously reported. Cho et al., Proc. Natl. Acad.Sci. USA 103:4946-4951 (2006). Following wounding, ear skin was treatedeither with topical tacrolimus or vehicle control for 4 weeks. Ears werethen harvested and fixed in 1% PFA overnight. The anterior and posteriorportions of the ear skin were then divided removing the cartilage andwhole mount staining for LYVE-1 and CD31 was performed. Tile-scan imageswere obtained using a confocal microscope (Leica Microsystems, Weitziar,Germany) and skin within 400 μm of the wound edge was analyzed usingMetamorph software (Molecular Devices, Sunnyvale, Calif.). Standardized(200 μm×200 μm) fields were analyzed for the number of branch pointspresent per unit area by two blinded reviewers.

Flow Cytometry

Flow cytometry was performed on peripheral blood samples as previouslyreported. Zampell et al., PLoS ONE 7:e49940 (2012). Briefly,erythrocytes were lysed with RBC lysis buffer (Ebioscience, San Diego,Calif.) followed by staining with fluorophore-conjugated antibodies(CD45, CD3, and CD4; all from Biolegend, San Diego, Calif.) and analysiswith a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, N.J.)using FlowJo software (Tree Star, Ashland, Oreg.).

Tacrolimus Blood Level by Mass Spectrometry

Blood levels of tacrolimus were measured using mass spectrometry in amodification of a previously reported method. Donaldson et al., Meth.Mol. Biol. 603:479-487 (2010). Briefly, whole blood was collected insodium EDTA-coated tubes (Terumo, Shibuya, Japan) and then analyzedusing a Thermo Scientific Aria TLX-2 turbulent flow chromatograph (TFC)coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer(Thermo Scientific, Franklin, Mass.). The TurboFlow column used was aCyclone P-50×0.5 mm while the analytical column was a Hypersil Gold C-18column, 3×50 mm. To whole blood (50 μL) was added 2004, of 0.2 mM ZnSO4containing ascomycin as an internal standard. Following a 30 minuteincubation and centrifugation, 50 μL of the supernatant was injectedinto the TFC system. The analytes were eluted through the column (0.75mL min⁻¹) with a gradient of water and methanol solutions containing 10mM ammonium formate and 0.1% formic acid. The analytical run time was4.5 minutes. The between-day imprecision of the assay was determined atthree concentrations over a period of 10 days. At concentrations of 3.3,12.6 and 31.9 ng mL⁻¹ the coefficients of variation were of 9.8, 7.0 and7.8%, respectively. The assay has a linear range from 0 to 40 ng mL⁻¹.

Analysis of Lymphatic Function

Tail volumes were calculated using the truncated cone formula aspreviously reported (Clavin et al., Am. J. Physiol. Heart Circ. Physiol.295:H2113-H2127 (2008)) and confirmed using histological measurements ofsoft tissue thickness of the skin/subcutaneous tissues in a standardizedmanner using Mirax Imaging Software (Carl Zeiss, Munich, Germany).

Lymphoscintigraphy was performed using our previously published methods.Avraham et al., Am. J. Pathol. 177:3202-3214 (2010). Briefly, 50 μl offiltered technetium-99m (^(99m)Tc) sulfur colloid (Nuclear DiagnosticProducts, Rockaway, N.J.) was injected in the distal tail. Images weretaken using an X-SPECT camera (Gamma Medica, Northridge, Calif.) andregion-of-interest analysis was performed to derive decay-adjustedcounts in the sacral lymph nodes and to calculate peak and rate of nodaluptake using ASIPro software (CTI Molecular Imaging, Knoxville, Tenn.).

Near infrared imaging (NIR) was performed using a modification ofpreviously published results. Tassenoy et al., Lymphat. Res. Biol.7:145-151 (2009). Briefly, 15 μl (0.15 mg mL⁻¹) indocyanine green (ICG,Sigma-Aldrich, Saint Louis, Mo.) was injected intradermally in the webspace of the dorsal hind limb and visualized using an EVOS EMCCD camera(Life Technologies, Carlsbad, Calif.) with a LED light source (CoolLED,Andover, United Kingdom). Static/video images were obtained using aZeiss V12 Stereolumar microscope (Caliper Life Sciences, Hopkinton,Mass.) and lymphatic pumping function was analyzed using Fiji software(NIH, Bethesda, Md.) by identifying a region-of-interest over thedominant collecting vessel of the leg and subtracting the backgroundfluorescent intensity plotted over time.

Histology and Immunostaining

Immunohistochemical staining was performed using our published methods.Clavin et al., Am. J. Physiol. Heart Circ. Physiol. 295:H2113-H2127(2008). Briefly, tissues were fixed in 4% paraformaldehyde at 4° C.,decalcified in 5% sodium EDTA (Santa Cruz Biotechnology, Dallas, Tex.),embedded in paraffin, and sectioned at 5 micrometers. Cut sections wererehydrated and heat-mediated antigen unmasking was performed using 90°C. sodium citrate (Sigma-Aldrich). Non-specific binding was blocked with2% BSA/20% animal serum. Tissues were incubated overnight with primaryantibody at 4° C. Primary antibodies used for immunohistochemical stainsincluded goat anti-mouse LYVE-1, rat anti-mouse CD45, rabbit anti-mouseCD4, and rat anti-mouse F4/80 (all from R&D, Minneapolis, Minn.), rabbitanti-mouse CD3 (from Dako, Carpinteria, Calif.), Cy3-conjugated mouseanti-aSMA (from Sigma-Aldrich), rabbit anti-human IFN-γ, rabbitanti-mouse TGF-β1, rabbit anti-mouse p-SMAD3, rabbit anti-mouse collagenI, rabbit-anti-mouse iNOS, and hamster-anti-mouse podoplanin, rabbitanti-mouse HMGB-1 and HSP-70 (all from ABCAM, Cambridge, Mass.).

Immunofluorescence staining was performed using AlexaFluorfluorophore-conjugated secondary antibodies (Life Technologies, Norwalk,Conn.). Images were scanned using Mirax imaging software (Carl Zeiss).Peri-lymphatic CD45⁺ and CD4⁺ cell counts were assessed by countingpositively stained cells within 50 μm of the most inflamed lymphaticvessel in each quadrant of the leg. Positively stained cells werecounted by two blinded reviewers in four randomly-selected, 40×high-power fields in a minimum of 4 fields per animal. Collagen Ideposition was quantified using Metamorph software (Molecular Devices,Sunnyvale, Calif.) in dermal areas of 5 μm cross-sections. This analysiswas confirmed using picrosirius red staining and scar index calculationas previously reported. Flanders et al., Am. J. Pathol. 163:2247-2257(2003). Bridging lymphatic vessels in mouse tails were counted in there-epithelialized surgical site in 4 different high-power fields pertail.

Sirius Red Staining

Paraffin sections of tail tissues were stained with picrosirius redstaining kit (Polysciences, Warrington, Pa.) according to themanufacturer's instructions. Images were obtained through polarizedlight on an Axiocam 2 microscope (Carl Zeiss) and the scar index wasquantified with Metamorph software by calculating the ratio ofred-orange:green-yellow fibers with higher numbers representingincreased scarring.

Real-Time PCR

RNA extraction was performed on tail skin using TRIZOL (Invitrogen, LifeTechnologies, Carlsbad, Calif.) according the manufacturer'srecommendations. RNA quality and quantity was assessed using an Agilentbio analyzer (Agilent Technologies, Inc; Santa Clara, Calif.). Theisolated RNA was converted to cDNA using a TaqMan reverse transcriptasekit (Roche, Branchburg, N.J.) and relative expression of gene expressionbetween groups was performed using delta-delta CT PCR analysis andnormalizing gene expression using GAPDH RNA amplification as previouslydescribed. Schmittgen et al., Nat. Protoc. 3:1101-1108 (2008). Relativeexpression was calculated using the formula: 2[−(Ct gene of interest—Ctendogenous control) sample A—(Ct gene of interest—Ct endogenous control)sample B)]. All samples were performed in triplicate. The primers usedfor the PCR targets of interest were for VEGF-C, TGF-β1, and IFN-γ(Applied Biosystems, Life Technologies, Carlsbad, Calif.).

In-Vivo Detection of ROS and Miles Assay for Vascular Permeability

In-vivo detection of ROS was performed by bioluminescent imaging ofNADPH oxidase as described by Han et al. (J. Vis. Exp. doi: 10.3791/3925(2012)). Briefly, mice were systemically injected with L-012 (ananalogue of luminol) (20 μg/g) dissolved in PBS at different time pointsafter PLND and luminescent signals representing ROS at the PLND surgerysite were imaged and quantified using IVIS spectrum 200 (XenogenCorporation). Miles assay for vascular permeability was performed asdescribed. Radu et al., J. Vis. Exp. doi: 10.3791/50062 (2013). Briefly,200 μl of 0.5% sterile Evans blue was injected via tail vein to 2 weekstacrolimus treated PLND mice. After 30 min PLND hind limbs were imagedto observe Evans blue leakage. Hind limbs were excised and incubated informamide for 48 hrs. at 55° C. to extract the Evans blue. ExtractedEvans blue was quantified by measuring absorbance at 610 nm.

Statistical Analysis

Data was analyzed and displayed using GraphPad Prism software (GraphPadSoftware, La Jolla, Calif.). Values are presented as mean±standarddeviation unless otherwise noted. Statistical significance was set atp<0.05, and differences between 2 groups was assessed with the Student'st-Test while multiple analyses were performed using ANOVA with post hoctests to compare within groups.

Conclusions

Because CD4⁺ T cells play a critical role in lymphedema pathology, thepurpose of this study was to evaluate the efficacy of topical tacrolimusfor the prevention and treatment of lymphedema. We used well a describedmouse tail model of lymphedema as well as a previously described modelof lymphatic injury resulting from popliteal lymph node dissection(PLND) to show that topical tacrolimus potently prevents development oflymphedema after lymphatic injury by decreasing chronic inflammatoryresponses, decreasing tissue and lymphatic fibrosis, increasingcollecting lymphatic pumping, and increasing collateral lymphatic vesselformation. These findings have important implications for the treatmentof lymphedema since previous experimental attempts have focusedprimarily on increasing lymphatic regeneration using exogenouslymphangiogenic growth factors.

We also found that tacrolimus potently decreases dermal and subcutaneousT cell infiltration and tissue fibrosis after lymphatic injury. Thesechanges prevent development of lymphedema and can reverse pathologicchanges once lymphedema is established. Treatment with tacrolimusincreases lymphatic function by increasing formation of collaterallymphatics and by increasing collecting lymphatic pumping frequency. Toour knowledge, this is the first targeted topical pharmacologic means ofpreventing and treating post-surgical lymphedema.

We found that tacrolimus was more effective when applied earlier,immediately after surgery, likely reflecting the fact that thistreatment did not require reversal of established pathology. Thisfinding is consistent with previous studies in other fibroproliferativedisorders such as hepatic fibrosis in which prevention is much moreeffective than reversal of histological changes. Friedman et al.,Hepatology 43:S82-S88 (2006).

Example 2 Treatment and Prevention of Lymphedema Using Pirfenidone

Pirfenidone Decreases Tail Lymphedema

Disruption of the superficial and deep lymphatics of the mouse tailresulted in almost 80% increase in tail volumes 2 weeks after surgery(FIG. 19(A), (B)). We have previously shown that swelling at this timepoint is due primarily to accumulation of interstitial fluid. Avraham etal., FASEB J. 27:1114-1126 (2013). Chronic lymphatic obstruction in thetail results in gradual replacement of interstitial fluid byfibroadipose tissues and accumulation of inflammatory cells over theensuing 4 weeks. Avraham et al., FASEB J. 27:1114-1126 (2013). Thesepathologic changes closely mirror clinical lymphedema and persist for atleast 8-10 additional weeks once lymphedema is established. Based onthis knowledge, we waited 7 weeks after surgery for lymphedema to becomeestablished and then initiated treatment with pirfenidone with an intentto treat these established soft tissue changes. We initially startedwith systemic treatment as most of the previous studies on pirfenidoneuse this route of administration for the treatment of fibrotic diseases.However, once we established pirfenidone as an effective treatment, wedeveloped a topical formulation, as local administration would bepreferred as to minimize any potential side effects due to systemictherapy. Treatment with both systemic and topical pirfenidone markedlydecreased gross tail swelling, tail volume, and soft tissue thickness ascompared with controls (FIG. 19(A)-(E); p<0.01 for tail volume andp<0.05 for thickness for both treatment groups).

Pirfenidone Increases Lymphatic Function in the Tail

Given the efficacy of pirfenidone in treating lymphedema in the tailmodel, we next sought to study if pirfenidone regulates lymphaticfunction after lymphatic injury. Using ⁹⁹Tc lymphoscintigraphy, atechnique in which a radiotracer is injected in the distal tail, wemeasured its uptake by the sacral lymph nodes over 90 minutes.Decay-adjusted uptake of the sacral lymph nodes in both systemic andtopical pirfenidone treated animals demonstrated an almost 4-foldincrease in ⁹⁹Tc uptake in pirfenidone-treated animals as compared withcontrols. In addition, both systemic and topically treated animalsdemonstrated an almost 4-fold increase in peak nodal uptake (FIG. 19(G);p<0.01 for both). Similarly, there was an increased in the rate ofuptake in both systemic and topically treated animals as indicated bythe increased slope of the decay-adjusted curve (FIG. 19 (H); p<0.01 andp<0.05, respectively).

Pirfenidone Decreases Inflammation and Fibrosis in the Tail AfterLymphatic Injury

Chronic inflammation is a histological hallmark of clinical lymphedemaand is characterized by increased accumulation of inflammatory cells,specifically T-helper cells. Avraham et al., FASEB J. 27:1114-1126(2013); Zampell et al., PLoS ONE 7:e49940 (2012); Ghanta et al., Am. J.Physiol. Heart Circ. Physiol. 308:H1065-1077 (2015); Olszewski et al.,Lymphology 23:23-33 (1990). Consistent with this, we found that bothsystemic and topically treated animals had markedly decreased numbers ofleukocytes infiltrating the dermis and subcutaneous fat as compared withcontrols. Inflammatory cells in lymphedematous tissues harvested fromcontrol animals were located in close proximity to the capillary andcollecting lymphatics, but were virtually absent in pirfenidone treatedmice. Similarly, we noted marked decreases in the numbers ofinfiltrating CD3⁺ cells, and CD4⁺ cells (FIG. 20(A), (D); p<0.001 forboth). Furthermore, we noted a significant decrease in accumulation ofIFN-γ protein, a T-helper (Th) 1 cell produced cytokine that haspreviously been found to be potently anti-lymphangiogenic (FIG. 20(J);p<0.01). Kataru et al., Immunity 34:96-107 (2011); Shao et al., J.Interferon Cytokine Res. 26:568-574 (2006).

In addition, we have previously shown macrophages to accumulate intissues distal to lymphatic injury. Zampell et al., PLoS ONE 7:e49940(2012). We analyzed the effect of pirfenidone on macrophage infiltrationsince they regulate both fibrosis (primarily through TGF-β1) andregulate lymphangiogenesis (via VEGF-C). We found no difference inF4/80⁺ cellular infiltration in lymphedematous tail tissues withpirfenidone treatment, both systemic and topical, compared to controls.Consistent with this, we found no difference in VEGF-C proteinaccumulation after treatment with pirfenidone (FIG. 20(K); p=NS). Takentogether, these findings show that following lymphatic injury,inflammatory cells, specifically T cells, accumulate in large numbers inclose proximity to skin/subcutaneous lymphatic vessels and that thisresponse is mitigated by systemic and topical pirfenidone treatment.

Patients with lymphedema have progressive soft tissue fibrosis and thedegree of fibrosis correlates with the severity of disease clinically.Tassenoy et al., Lymphat. Res. Biol. 7:145-151 (2009). Consistent withother fibrotic disorders, we have previously shown TGF-β1 to be acritical pro-fibrotic growth factor in lymphedema. Avraham et al., Am.J. Pathol. 177:3202-3214 (2010). In addition, we have shown TGF-β1 tohave direct anti-lymphangiogenic effect on LECs. Clavin et al., Am. J.Physiol. Heart Circ. Physiol. 295:H2113-H2127 (2008). Therefore, weanalyzed infiltration of TGF-β1⁺ cells and cellular expression of itsactivated downstream signaling molecule, pSMAD3. In both systemic andtopical pirfenidone treatment groups we found markedly decreasedaccumulation of both TGF-β1⁺ cells and pSMAD3⁺ cells as compared tocontrols (FIG. 20(B), (C), (E), (F); p<0.001 for all). Consistent withthis, we found decreased accumulation of TGF-β1 protein accumulationafter treatment with pirfenidone (FIG. 20(I); p<0.05). This correlatedwith significantly decreased dermal and subcutaneous type I collagendeposition in both treatment groups as compared with control mice (FIG.19(D), (F); p<0.001 for systemic and p<0.05 for topical).

Since structural changes in collecting lymphatic vessels (hypertrophy ofsmooth muscle, thick layers of type I collagen, constriction of thelumen) have been described in human patients with lymphedema, weexamined the collecting lymphatic vessels of our animals for similarchanges. Mihara et al., PLoS One 7:e41126 (2012). Consistent with thesehuman studies, collecting lymphatics (α-SMA⁺/podoplanin⁺) of controlmice were surrounded by thick layers of type I collagen whilepirfenidone-treated animals had essentially normal lymphatic vessels(FIG. 20(H)).

Pirfenidone Increases Lymphatic Function in the Hind Limb

In order to understand the mechanisms by which pirfenidone increaseslymphatic function, we analyzed dermal backflow in initial lymphaticsand collecting lymphatic pumping capacity using a mouse hind limb PLNDmodel. Blum et al., Breast Cancer Res. Treat. 139:81-86 (2013). UsingNIR lymphangiography 4 weeks after PLND, we found that animals treatedwith 2 weeks of systemic pirfenidone had markedly less dermal backflowand capillary vessel leakage as compared with vehicle-treated controls(FIG. 18(A), white arrow). In addition, pirfenidone-treated animals hadsignificantly increased rate of collecting lymphatic vessel contractionas compared with controls (2-fold increase; FIG. 18(A), (B); p<0.0015).This response, similar to our findings with the tail model, correlatedwith a significant decrease in perilymphatic infiltration ofinflammatory cells in mice treated with pirfenidone (FIG. 18(E) (upperpanels), FIG. 18(F); p<0.01). In addition, treatment with pirfenidoneresulted in a significant decrease in perilymphatic expression of iNOSby inflammatory cells (FIG. 18(E) (lower panels), FIG. 18(G); p<0.01).This is a critical finding as increased peri-lymphatic iNOSaccumulation, in the setting of inflammation, has been shown to disruptnormal nitric oxide gradients that are critical for normal, coordinatedlymphatic vessel contractility. Liao et al., Proc. Natl. Acad. Sci. USA108:18784-18789 (2011). This provides an important mechanism as to howpirfenidone improves lymphatic vessel contractility.

Pirfenidone Increases Collateral Lymphatic Vessel Formation

Since T cells, specifically Th1 and Th2 cytokines, are known to potentlyinhibit lymph node lymphangiogenesis and inflammatory lymphangiogenesisduring wound repair, we next sought to determine if treatment withpirfenidone increases the formation of collateral lymphatics. Kataru etal., Immunity 34:96-107 (2011); Clavin et al., Am. J. Physiol. HeartCirc. Physiol. 295:H2113-H2127 (2008); Zampell et al., Am. J. Physiol.Cell Physiol. 302:C392-C404 (2012); Savetsky et al., PLoS One10:e0126908 (2015). Indeed, histological analysis and identification oflymphatics using LYVE-1 immunofluorescent staining demonstrated a markedincrease in lymphatic vessels density in both the tail and hind limb(FIG. 18(D), (E); p<0.001). This lymphangiogenic response appeared to beindependent of VEGF-C expression since we noted no differences in VEGF-Cprotein analysis in pirfenidone-treated lymphedematous tails as comparedto controls whereas there was a profound decrease inanti-lymphangiogenic T cell cytokines such as IFN-γ and TGF-β1 (FIG.20(E), (I), (K); p<0.05 for TGF-β1, p<0.01 for IFN-γ, and p=NS forVEGF-C).

TGF-β1 Immunotherapy with Pirfenidone does not Further Improve LymphaticFunction

Given that the major mechanism of action of pirfenidone is blockade ofTGF-β1 activity, we compared the effect of pirfenidone treatment withTGF-β1 immunotherapy after PLND in separate studies. Schaefer et al.,Eur. Respir. Rev. 20:85-97 (2011). Using MR imaging, compared to isotypecontrols, we found that TGF-β1 immunotherapy alone as well as thecombination of TGF-β1 immunotherapy and pirfenidone had decreased dermalbackflow, and significantly increased rate of collecting lymphaticvessel contraction. More importantly, there was no added benefit withcombination therapy compared to immunotherapy alone, indicating that wewere maximally inhibiting TGF-β1 at the doses we utilized (FIG.21(A)-(C); p=NS). Similarly, using flow cytometry, compared to isotypecontrols, we found that TGF-β1 immunotherapy alone as well as thecombination of TGF-β1 immunotherapy and pirfenidone had significantlydecreased tissue accumulation of T-helper cells distal to the lymphaticinjury whereas there was no additional with combination therapy comparedto immunotherapy alone.

TGF-β1 KO From T Cells Decreases Tail Lymphedema, Improves LymphaticFunction and Decreases Inflammation and Fibrosis in the Tail AfterLymphatic Injury

To determine the cellular source of TGF-β1 in the setting of lymphedema,we developed transgenic mice with selective KO of TGF-β1 production fromT cells and myelocytes. Phenotypic confirmation of these transgenic micewas confirmed using polymerase chain reaction (PCR) that showedsignificant decreased TGF-β1 expression from isolated T and myeloidcells from T Cell^(cre) and Myeloid^(cre) mice, respectively, ascompared to control mice (FIG. 21(A); p<0.05 for both). Tail surgerieswere performed and analysis was performed 6 weeks after surgery. TCell^(cre) mice had markedly decreased gross tail swelling, tail volume,and fibroadipose thickness as compared to Myeloid^(cre) and control mice(FIG. 21(B)-(D); p<0.01 for tail volume and p<0.05 for thickness).Analysis of ⁹⁹Tc lymphoscintigraphy demonstrated an almost 4-foldincrease in decay-adjusted uptake of the tracer in the sacral lymphnodes of T Cell^(cre) mice as compared to Myeloid^(cre) and controlmice. Similarly, there was an increase in peak nodal uptake as well asthe rate of tracer uptake in the T Cell^(cre) mice as compared toMyeloid^(cre) and control mice (FIGS. 21(E), (F); p<0.05 and p<0.01,respectively).

Previously, we have shown that TGF-β1 immunotherapy in our tail model oflymphedema resulted in decreased inflammation, specifically T-helpercell tissue infiltration, in addition to decreased fibrosis. Avraham etal., Am. J. Pathol. 177:3202-3214 (2010). Interestingly, T Cell^(cre)mice had markedly decreased numbers of T-helper cells infiltrating thedermis and subcutaneous fat as compared with Myeloid^(cre) and controlmice (FIG. 22 (A), (B); p<0.01). Furthermore, similar to our findings inthe pirfenidone treated animals, we noted a significant decrease inaccumulation of IFN-γ protein (FIG. 22(D); p<0.01). Analysis of TGF-β1⁺cells and protein accumulation as well as cellular expression of itsactivated downstream signaling molecule, pSMAD3, was found to bemarkedly decreased in T Cell^(cre) mice as compared with Myeloid^(cre)and control mice (FIG. 22(A), (C), (E); p<0.01 for pSMAD3 and p<0.05 forTGF-β1 protein). We found no difference in F4/80⁺ cells or VEGF-Cprotein accumulation between all groups (FIG. 22(F); p=NS). Thiscorrelated with significantly decreased dermal and subcutaneous type Icollagen deposition in T Cell^(cre) mice as compared with Myeloid' andcontrol mice (FIG. 21(D); p<0.05). While there appeared to be a trend indecreased inflammation, fibrosis, and anti-lymphangiogenic cytokineexpression in Myeloid^(cre) mice it did not achieve significance. Thesefindings are important because they indicate that the major cellularsource of TGF-β1 in lymphedema is T cells.

Impaired LEC TGF-β1 Responsiveness Had No Change in Tail Lymphedema,Inflammation, or Fibrosis in the Tail After Lymphatic Injury

To analyze the direct effects of TGF-β1 on LECs in the setting oflymphedema and apply our previous in vitro findings to an in vivo model,we performed tail surgeries on FLT4^(Cre) mice and analysis wasperformed 6 weeks after surgery. Avraham et al., Plast. Reconstr. Surg.124:438-450 (2009). Phenotypic confirmation of these transgenic mice wasconfirmed with staining on lymph nodes for LYVE-1 and pSMAD3. FLT4^(Cre)mice had no detectable pSMAD3 staining on LECs, indicating LECunresponsiveness to TGF-β1 (FIG. 23(A), white arrows indicate pSMAD3⁻LECs). FLT4^(re) mice had no differences in gross tail swelling, tailvolume, and fibroadipose thickness as compared to control mice (FIG.23(C)-(E), (G); p=NS). Interestingly and similar to our previous invitro findings, we found increased lymphatic vessel density in thebridging portion of the wounds (FIG. 23(H); p<0.01). Clavin et al., Am.J. Physiol. Heart Circ. Physiol. 295:H2113-H2127 (2008). The bridgingportion of the wound is the area where all dermal lymphatic vessels wereremoved with surgery. Therefore, this indicates improvedlymphangiogenesis as a result of LEC unresponsiveness to TGF-β1 in thesetting of lymphedema.

Analysis of the inflammatory infiltrating cells and cytokineaccumulation between FLT4^(Cre) and control mice revealed no differencesin CD4⁺ cells and IFN-γ protein accumulation (p=NS for both), F4/80⁺cells or VEGF-C protein accumulation (p=NS), TGF-β1⁺ cells and proteinaccumulation (FIG. 23(B); p=NS), as well as cellular expression of itsactivated downstream signaling molecule, pSMAD3 (FIG. 23(G); p=NS).Similarly, collagen I deposition was not significantly different betweenFLT4^(Cre) and control mice (FIG. 23(F), (G); p=NS). These findingssuggest that the main effect of TGF-β1 in promoting inflammation,fibrosis and ultimately lymphatic dysfunction is in the extracellularmatrix while its direct anti-lymphangiogenic plays a relatively minorrole in the setting of lymphedema.

Methods

Study Design

Our hypothesis was that lymphedema could be treated by TGF-β1inhibition, thereby decreasing both inflammation and fibrosis. Weexplored different aspects of this hypothesis in several differentanimal models to allow us to thoroughly assess swelling, inflammation,fibrosis, lymphatic vessel function, and lymphangiogenesis after injury.Adult female (10-14 week old) C57BL/6J mice (Jackson Laboratories, BarHarbor, Me.) were maintained in light- and temperature-controlledpathogen free environments, and fed ad libitum. All studies wereapproved by the Institutional Animal Care and Use Committee (IACUC) atMemorial Sloan Kettering Cancer Center. After undergoing tail surgery,animals were excluded from the experiment if they underwent distal tailnecrosis. This assessment was made before animals were randomized totreatment or control groups. Each experiment was performed using aminimum of 6-8 animals and assays were performed in triplicate. Allcounts were performed by reviewers blinded to the intervention.

Animal Models and Treatments

Animals underwent lymphatic ablation using a well-described mouse tailmodel of lymphedema in which the superficial and deep lymphatic systemsof the tail are excised through a 2 mm circumferential skin excision inthe mid-portion of the tail. Clavin et al., Am. J. Physiol. Heart Circ.Physiol. 295:H2113-H2127 (2008); Avraham et al., Am. J. Pathol.177:3202-3214 (2010); Rutkowski et al., Microvasc. Res. 72:161-271(2006); Tabibiazar et al., PLoS Med. 3:e254 (2006). Our group and othershave previously shown that this model results in sustained lymphedema ofthe distal tail, severe impairment in lymphatic function, andhistological features of clinical lymphedema (e.g., chronicinflammation, adipose deposition, fibrosis) for at least 10 weekspostoperatively. Clavin et al., Am. J. Physiol. Heart Circ. Physiol.295:H2113-H2127 (2008); Avraham et al., Am. J. Pathol. 177:3202-3214(2010); Rutkowski et al., Microvasc. Res. 72:161-271 (2006); Tabibiazaret al., PLoS Med. 3:e254 (2006). Seven weeks after surgery, whenlymphedema was established, animals were randomized to experimental(pirfenidone) or control groups and treated once daily for 3 weeksfollowed by analysis as outlined below.

The tail model is useful for analyzing histological tissue changes(i.e., fibrosis and adipose deposition); however, due to the smallcaliber of the collecting vessels this model, is not ideal for analyzinglymphatic pumping function. Therefore, to determine the efficacy ofpirfenidone in restoring lymphatic pumping capacity of collectinglymphatics and to analyze the effect of this treatment on lymphaticproliferation in the tissues distal to the zone of lymphatic injury, weperformed popliteal lymph node dissections (PLND) as previouslydescribed. Blum et al., Breast Cancer Res. Treat. 139:81-86 (2013).Briefly, the lymphatics were visualized after injection of 50 μl of 3%Evans Blue into the hind paw. The collecting lymphatics in the poplitealregion, together with the popliteal lymph nodes, were excised. Two weeksfollowing surgery, animals were randomized to treatment with systemicpirfenidone or control once daily for 2 weeks followed by analysis.

In systemic experiments, mice were treated orally daily with eitherpirfenidone (Cayman Chemical, Ann Arbor, Mich.) at a dose of 400 mg/kgdissolved in 10% DMSO/0.5% carboxymethycellulose (CMC) in PBS or withvehicle (10% DMSO/0.5% CMC in PBS). This dose was determined based onprevious studies that showed an effective treatment regimen in variousmodels of fibrosis. Oku et al., Eur. J. Pharmacol. 590:400-408 (2008);Kakugawa et al., Eur. Respir. J. 24:57-65 (2004); Tanaka et al., Chest142:1011-1019 (2012). A topical formulation of pirfenidone was developedin collaboration with the Research Pharmacy Core Facility at MemorialSloan Kettering Cancer Center. In these experiments, mice were treateddaily with topical pirfenidone (Cayman Chemical, Ann Arbor, Mich.) 1mg/ml in 41% petrolatum while control animals received vehicle alone(41% petrolatum).

To investigate the cellular source of TGF-β1 in lymphedema we developednon-inducible transgenic mice with selective knock out of TGF-β1production from T cells and myelocytes. From Jackson Laboratories (BarHarbor, Me.), we purchased B6.Cg-Tg(Lck-cre)548Jxm/J that express Creunder the control of the Lck (lymphocyte protein tyrosine kinase)promoter, enabling thymocyte-specific excision of loxP-flanked sequencesof interest. The Lck gene is primarily expressed by T lymphocytes whereit phosphorylates tyrosine residues of proteins involved withintracellular signaling pathways. Additionally, we purchased fromJackson Laboratories (Bar Harbor, Me.) a B6.129P2-Lyz2tml (cre)Ifo/Jtransgenic strain with the LysMcre knock-in allele that has anuclear-localized Cre recombinase inserted into the first coding ATG ofthe lysozyme 2 gene (Lyz2); both abolishing endogenous Lyz2 genefunction and placing NLS-Cre expression under the control of theendogenous Lyz2 promoter/enhancer elements. Each of these transgenicmice was crossed with Tgfbl^(tm2.1Doe)/J mutant mice (JacksonLaboratories, Bar Harbor, Me.) that harbor loxP sites flanking exon 6 ofthe TGF-β1 gene. As a result, Cre-mediated recombination results indeletion of the targeted gene (TGF-β31) in T lymphocytes and myeloidcell lineage (including monocytes, mature macrophages, andgranulocytes).

To investigate the direct effects of TGF-β1 on LECs, we developed aninducible transgenic mouse with a non-functional TGF-β receptor on LECs.We used an FLT4cre mouse (a gift of Dr. Sagrario Ortega) where the Flt4promoter of VEGFR-3 in these mice is under the control of estrogenreceptor type 2 (ER2) and is highly expressed by all LECs in adult mice.Martinez-Corral et al., Proc. Natl. Acad. Sci. USA 109:6223-6228 (2012).We crossed FLT4cre mice with B6; 129-Tgfbr2tmlKarl/J (JacksonLaboratories, Bar Harbor, Me.) mutant mice that possesses loxP sitesflanking exon 4 of the transforming growth factor, beta receptor II. Creexpression was induced in adult female FLT4cre mice using tamoxifen (300mg/kg/day subcutaneously for 5 days).

For all transgenic mice, gene expression of both transgenes wasconfirmed by genotyping (Transnetyx, Memphis, Tenn.) and doublehomozygous mice were backcrossed for 6 generations to ensureconsistency. In addition, our lab performed confirmatory phenotypicstudies for all of our newly developed transgenic models with geneexpression using PCR, protein quantification using enzyme-linkedimmunosorbent assays (ELISA), and histologic staining (see below).

Tail Volumes, Lymphoscintigraphy, and Histological Analysis

Tail volumes were analyzed using multiple digital caliper tailcircumference measurements distal to the zone of lymphatic injury andcalculated using the truncated cone formula as previously described.Clavin et al., Am. J. Physiol. Heart Circ. Physiol. 295:H2113-H2127(2008). Lymphoscintigraphy was also performed as previously described toquantify lymphatic flow to the sacral lymph nodes by injecting 50 μl offiltered technetium (Tc^(99 m)) sulfur colloid into the distal tail.Avraham et al., FASEB J. 27:1114-1126 (2013). Decay-adjusted uptake wasrecorded in the sacral lymph nodes using an X-SPECT camera (GammaMedica, Northridge, Calif.) and region-of-interest analysis wasperformed using ASIPro software (CTI Molecular Imaging, Knoxville,Tenn.). Clavin et al., Am. J. Physiol. Heart Circ. Physiol.295:H2113-H2127 (2008).

For histological and immunohistochemical analysis, tail sections wereharvested, briefly fixed in 4% ice cold paraformaldehyde (Sigma-Aldrich,St. Louis, Mo.), decalcified using 5% ethylenediaminetetraacetic acid(EDTA; Santa Cruz, Santa Cruz, Calif.), and embedded in paraffin.Hematoxylin and eosin sections were prepared using standard techniquesand subcutaneous tissue thickness was quantified in standardizedhistological cross-sections by measuring the thickness of the skin andsoft tissues in four quadrants of the tail by two blinded reviewers in aminimum of 6 animals per group.

Immunohistochemical staining was performed according to our establishedtechniques. Avraham et al., Am. J. Pathol. 177:3202-3214 (2010).Paraffin-embedded tissues were rehydrated and antigen unmasking wasperformed using boiling sodium citrate (Sigma-Aldrich, St. Louis, Mo.)followed by quenching of endogenous peroxidase activity with 2% BSA/20%animal serum. Tissues were incubated with primary antibody overnight at4° C. Primary antibodies used for immunohistochemical stains included,LYVE-1, CD45, and CD4 (all from R&D, Minneapolis, Minn.), F4/80, TGF-β1,pSMAD3, Podoplanin (all from Abcam, Cambridge, Mass.), alpha-SMA(Sigma-Aldrich, St. Louis, Mo.), iNOS (BD biosciences, San Jose,Calif.), and CD3 (Dako North America, Inc. Carpinteria, Calif.). Allsecondary antibodies were obtained from Vector Laboratories. Slides wereanalyzed after being scanned using a Mirax slide scanner (Zeiss, Munich,Germany). Type I collagen immunohistochemistry was performed using anantibody to mouse type I collagen (Abcam, Cambridge, Mass.) andquantified as a ratio of the area of positively stained dermis within afixed threshold to total tissue area using Metamorph Offline software(Molecular Devices, Sunnyvale, Calif.). Cell counts were performed onhigh-powered sections, with a minimum of 4-6 animals per group and 4-5HPF/animal by two blinded reviewers.

Protein Analysis

Tail tissues for protein analysis was harvested 1.5 cm distal to thelymphatic injury, flash frozen, crushed and extracted with tissueextraction protein reagent (ThermoFisher Scientific, Waltham, Mass.)mixed with phosphatase and protease inhibitor (Sigma-Aldrich, St. Louis,Mo.). 20-30 mg of protein from samples (n=3-5 animals/group) wasanalyzed by ELISA to quantify TGF-β1, interferon-gamma (IFN-γ), andvascular endothelial growth factor-C (VEGF-C) according to themanufacturer's protocol (eBioscience, San Diego, Calif.). Allexperiments were performed in duplicate.

In Vivo Imaging of Lymphatic Vessel Function

To assess the contractility of hind limb collecting lymphatic vessels,videos were recorded with near infrared imaging (NIR) after 15 μl of0.15 mg/ml indocyanine green (ICG) (Sigma-Aldrich, Saint Louis, Mo.) wasinjected intradermally in the dorsal aspect of the hind foot. Twentyminutes was allowed after injection to allow uptake into the collectingvessels. The animals were then imaged using a custom-made EVOS EMCCDcamera (Life Technologies, Carlsbad, Calif.) and a LED light source(CoolLED, Andover, UK). Video images were obtained using a Zeiss V12Stereolumar microscope (Caliper Life Sciences, Hopkinton, Mass.). Imageswere obtained every eight seconds for 30 minutes. Lymphatic pumpingfunction was analyzed using Fiji software (a free open source dataanalysis tool developed by the National Institutes of Health, Bethesda,Md.). A region-of-interest was selected over the dominant collectingvessel and the noise-subtracted fluorescent intensity was plotted overtime. In order to evaluate intrinsic pumping function, the initial tenminutes of each video was excluded due to lymphatic stimulation frompositioning, and only the final 20 minutes to each video were analyzed.The pumping function was quantified in pulsations per minute.

Gene Expression PCR

To confirm the phenotype of knock out of TGF-β1 from T cells andmyelocytes, spleens were harvested from these mice. T cells wereisolated from spleens of T Cell^(cre) transgenic mice using positiveselection CD3 magnetic beads (Miltenyi Biotec, Cambridge, Mass.) as perthe manufacture's recommendations. Similarly, myeloid cells wereisolated from spleens of Myeloid^(cre) transgenic mice using positiveselection CD11b magnetic beads (Miltenyi Biotec, Cambridge, Mass.) asper the manufacture's recommendations. Isolated cells were then placedin TRIzol. RNA was isolated using a standard TRIzol extractionprocedure. Chomczynski et al. Anal. Biochem. 162:156-159 (1987); Ribaudoet al., Curr. Protocols Immunol., (Coligan et al. eds.) Chapter 10, Unit10 11 (2001). Reverse transcription was performed using TaqMan ReverseTranscription reagents (Applied Biosystems, Foster City, Calif.)followed by quantitative reverse transcriptase polymerase chain reaction(RT-PCR) using TaqMan Universal Mastermix (Applied Biosystems) andLightCycler thermocycler (Roche Diagnostics, Indianapolis, Ind.).TGF-β1expression levels were normalized to GAPDH. Experiments wereperformed in triplicate.

Modulation of TGF-β1 Activity

Because previous studies have suggested that a major mechanism of actionof pirfenidone is blockade of TGF-β1 activity, we compared the effect ofpirfenidone treatment with TGF-β1 immunotherapy after PLND in separatestudies. Schaefer et al., Eur. Respir. Rev. 20:85-97 (2011).Importantly, we have previously shown that monoclonal antibodiesdirected against TGF-β1 are not only effective in neutralizing TGF-β1biologic activity but that this treatment markedly decreases lymphedemaand improves lymphatic function. Avraham et al., Am. J. Pathol.177:3202-3214 (2010). Animals underwent PLND as outlined above and twoweeks after surgery were randomized to treatment with either TGF-βmonoclonal mouse neutralizing antibody (TGFmab; clone 1D11; Bio-x-cell,West Lebanon, N.H.) alone or pirfenidone plus TGFmab at a dose of 5mg/kg diluted in 150 μl of PBS delivered intraperitoneally three timesper week. Ruzek et al., Immunopharmacol. Immunotoxicol. 25:235-257(2003). Control animals were treated with either vehicle control forpirfenidone or non-specific isotype antibodies deliveredintraperitoneally at the same schedule as TGFmab.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism (GraphPadSoftware, Inc., San Diego, Calif.) software. The Student's T-test wasused to compare differences between two groups. Analysis betweenmultiple time points (lymphoscintigraphy) was performed using a two-wayANOVA with post hoc tests to compare individual groups. Descriptiveanalysis and graphical methods were used to analyze and summarizeresults. Data is presented as mean±standard deviation unless otherwisenoted, with p<0.05 considered significant.

Conclusions

One purpose of this study was to analyze the efficacy of pirfenidone onthe treatment of lymphedema in preclinical mouse models of lymphedemaand lymphatic injury. Using two different mouse models, we show thatboth systemic and topical pirfenidone treat established lymphedema,markedly decreasing fibrosis and improving lymphatic function. Treatmentwith pirfenidone decreases chronic inflammatory reactions and markedlyincreases lymphatic collecting vessel pumping capacity afterlymphadenectomy. In addition, pirfenidone was highly effective inpreventing fibrosis after lymphatic injury, and it is likely that thisresponse is secondary to inhibition of TGF-β1. Using TGF-β1immunotherapy in our PLND model, we showed pirfenidone's major effect tobe TGF-β1 inhibition and that maximal inhibition of TGF-β1 was achievedat the doses we utilized. Furthermore, using our tail model oflymphedema in our mice, we show that T cells, specifically CD4+ cells,are the predominant source of TGF-β1 in the setting of lymphedema. Inaddition, we showed that the lack of TGF-β1 from T cells reducedlymphedema-induced chronic inflammation such as CD4⁺ cells along withTh1 and Th2 cytokines.

Example 3 Treatment and Prevention of Lymphedema Using Teriflunomide

To test the efficacy of teriflunomide in preventing lymphaticdysfunction after lymphatic injury, we used a well described mouse modelof popliteal lymph node dissection (PLND) in which popliteal lymph nodesare removed using a small skin incision. In order to test the efficacyof this treatment in treating established lymphedema, we used a mousetail model of lymphedema in which the superficial and deep lymphaticsystem of the tail is disrupted and animals develop histological changesthat are consistent with clinical disease.

Animals were randomized to either the PLND or tail lymphedema groups and2 weeks after surgery were treated with a topical formulation ofteriflunomide (27 mg/ml; Tocris Bioscience, Minneapolis, Minn.) orvehicle control (Aquaphor/glycerin ointment) once daily for 2-4 weeks (2weeks after PLND; 4 weeks after tail lymphedema). Our topicalformulation was developed in collaboration with the Research PharmacyCore Facility at Memorial Sloan Kettering Cancer Center. Mice were thensacrificed and lymphatic function, fibrosis, lymphangiogenesis were allassessed using standard assays.

Treatment of mice with topical teriflunomide after tail lymphaticablation markedly decreased lymphedema and fibroadipose deposition, thehistological hallmark of the disease (FIG. 24(A)-(D)). While controlmice had obvious swelling and fibrosis of the tail (fixed,“J-configuration” resulting from asymmetric collagen deposition),teriflunomide treated mice had essentially normal appearing tails 6weeks after lymphatic injury (FIG. 24(A)). These gross changescorresponded to a nearly 6-fold decrease in tail volume in teriflunomidetreated mice (FIG. 24(B)). Histological cross sections of control micetails showed significant accumulation of fibroadipose tissues; incontrast, teriflunomide treated mice had minimal adipose tissuedeposition (FIG. 24(C), (D)). This finding was confirmed with type Icollagen immunoflorescent staining demonstrating encasement ofsuperficial lymphatics by collagen fibers in control animals and markeddecreases in collagen deposition in teriflunomide treated animals 6weeks after injury (FIG. 25(A), (B)). In addition, teriflunomidedecreased proliferation of alpha smooth muscle cells surroundingcollecting lymphatics thus maintaining a more normal anatomicalconfiguration of these vessels (FIG. 25(C), (D)).

To determine if teriflunomide therapy decreased infiltration of CD4+cells, we next analyzed tissue sections from control and treated miceusing immunoflorescent antibodies targeting CD4 (FIG. 26(A), (B)). Thisanalysis demonstrated a marked decrease in CD4+ cell infiltration tailtissues harvested from teriflunomide treated animals as compared withcontrols. In fact, teriflunomide therapy decreased the number ofinfiltrating CD4+ cells by more than 8-fold (P<0.001).

Recent studies have shown that CD4+ cells produce potentanti-lymphangiogenic cytokines including interferon gamma, interluekin-4(IL4), and IL13. Consistent with this, we found that treatment ofanimals that underwent PLND with teriflunomide markedly increased (4.5fold; p<0.001) formation of collateral lymphatic vessels bypassing thezone of injury as assessed using indocyanine green (ICG) near infra-redimaging (FIG. 27(A)). Similarly, treatment of mice with teriflunomideafter tail lymphatic ablation resulted in a significant increase innewly formed lymphatics that crossed the zone of injury as compared withcontrols (FIG. 27(B)). The regeneration of collateral lymphatics interiflunomide animals significantly decreased leakiness of lymphaticvessels in the dermis enabling greater amounts of interstitial fluid tobe propagated proximally (FIG. 28).

The newly formed lymphatics and decreased pathological changes inexisting lymphatics of teriflunomide treated mice translated to markedlyimproved lymphatic function as analyzed by migration of dendritic cells(DCs). DCs migrate from the peripheral tissues via lymphatic vessels toregional lymph nodes to present antigens and promote adaptive immuneresponses. Analysis of DC trafficking in teriflunomide animals afterPLND using a standard assay (FITC painting) demonstrated a more than5-fold increase in the number of DCs that had trafficked to the inguinallymph node (the next lymph node in the chain following the popliteallymph node) as compared with controls (FIG. 29(A), (B)). Because DCsonly traffic via the lymphatics, this finding provides substantialevidence that teriflunomide increases lymphatic function.

Collecting lymphatic vessels propagate lymph proximally using activecontraction by surrounding smooth muscle cells and one way valves toprevent backflow. Consistent with our finding that teriflunomideincreases lymphatic transport function and decreasesfibrosis/proliferation of alpha smooth muscle cells surroundinglymphatic vessels, we found that this treatment also markedly increasedcollecting lymphatic pumping (FIG. 30(A), (B)). Teriflunomide treatmentafter PLND nearly doubled the frequency of the main hind limb collectinglymphatics thereby increasing proximal propagation of lymphatic fluid.

We describe for the first time the use of teriflunomide for preventionand treatment of lymphedema. Our findings show that treatment withteriflunomide after lymphatic injury substantially decreases lymphedemaand fibroadipose tissue deposition. In addition, we show that thiseffect correlates with decreased infiltration of CD4+ T cells, increasedformation of collateral lymphatics, decreased lymphatic leakiness, andimproved lymphatic function. These findings are novel since they providea targeted therapy for lymphedema, a disease that has previously beentreated only with palliative interventions.

Example 4 Treatment and Prevention of Lymphedema Using Captopril

As described above, we tested the efficacy of captopril in preventinglymphatic dysfunction after lymphatic injury using the PLND model, andin treating established lymphedema using the mouse tail model. Animalswere treated with a topical formulation of captopril (5%) or vehiclecontrol (petroleum jelly or Aquaphor/glycerin ointment) once daily for2-4 weeks (2 weeks after PLND; 4 weeks after tail lymphedema). Mice werethen sacrificed and lymphatic function, fibrosis, and lymphangiogenesiswere all assessed using standard assays. Treatment with topicalcaptopril resulted in improved lymphatic function in the PLND model andin decreased lymphedema in the tail model. Results are shown in FIGS.31-53.

Example 5 Treatment and Prevention of Lymphedema Using Drug Combinations

Using the PLND and mouse tail models, as described in Examples 1-4,animals are administered a topical formulation comprising a combinationof anti-fibrosis drugs or a vehicle control. Treatment compositions areshown in Table 1.

TABLE 1 Drug Combinations for Treatment and Prevention of LymphedemaComposition Components Conc. A1 tacrolimus 0.1%   pirfenidone 1 mg/ml A2tacrolimus 0.05%   pirfenidone 0.5 mg/ml B1 tacrolimus 0.1%  pirfenidone 1 mg/ml teriflunomide 27 mg/ml B2 tacrolimus 0.05%  pirfenidone 0.5 mg/ml teriflunomide 13.5 mg/ml C1 teriflunomide 27 mg/mlpirfenidone 1 mg/ml C2 teriflunomide 13.5 mg/ml pirfenidone 0.5 mg/ml D1tacrolimus 0.1%   captopril 5% teriflunomide 27 mg/ml D2 tacrolimus0.05%   captopril 2.5%   teriflunomide 13.5 mg/ml E1 teriflunomide 27mg/ml captopril 5% E2 teriflunomide 13.5 mg/ml captopril 2.5%   F1tacrolimus 0.1%   captopril 5% F2 tacrolimus 0.05%   captopril 2.5%   G1tacrolimus 0.1%   pirfenidone 1 mg/ml leflunomide 10%  G2 tacrolimus0.05%   pirfenidone 0.5 mg/ml leflunomide 5% H1 leflunomide 10% pirfenidone 1 mg/ml H2 leflunomide 5% pirfenidone 0.5 mg/ml I1tacrolimus 0.1%   captopril 5% leflunomide 10%  I2 tacrolimus 0.05%  captopril 2.5%   leflunomide 5% J1 leflunomide 10%  captopril 5% J2leflunomide 5% captopril 2.5%  

Mice are treated once daily for 2-4 weeks (2 weeks after PLND; 4 weeksafter tail lymphedema). Mice are then sacrificed and lymphatic function,fibrosis, lymphangiogenesis are assessed using standard assays. In thePLND model, treatment with a combination of topical anti-fibrosis drugsresults in improved lymphatic function compared to treatment with asingle anti-fibrosis drug. Likewise, in the tail model, treatment with acombination of topical anti-fibrosis drugs results in decreasedlymphedema compared to treatment with a single anti-fibrosis drug. Inaddition to being more effective, the combination produces synergisticeffects, such that the effective dose of each drug administered in thecombination is lower than the effective dose of each drug administeredalone.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance. The present invention is further described by the followingclaims.

The invention claimed is:
 1. A method of treating lymphedema, the methodcomprising administering to a subject with lymphedema a pharmaceuticalcomposition comprising an effective amount of pirfenidone.
 2. The methodof claim 1, wherein the pharmaceutical composition is administeredtopically.
 3. The method of claim 2, wherein the pharmaceuticalcomposition is in a form selected from the group consisting of anointment, a cream, a lotion, a paste, a gel, a mousse, a foam, alacquer, a suspension, a liquid, and a spray.
 4. The method of claim 3,wherein the pharmaceutical composition is in the form of an ointment. 5.The method of claim 2, wherein the pharmaceutical composition isadministered at least once a day.
 6. The method of claim 5, wherein thepharmaceutical composition is administered at least twice a day.
 7. Themethod of claim 1, wherein the pharmaceutical composition isadministered orally.
 8. The method of claim 7, wherein thepharmaceutical composition is in a form selected from the groupconsisting of a tablet, a capsule, a liquid, a solution, a softgel, asuspension, an emulsion, a syrup, and an elixir.
 9. The method of claim8, wherein the pharmaceutical composition is in the form of a tablet ora capsule.
 10. The method of claim 7, wherein the pharmaceuticalcomposition is administered at least once a day.
 11. The method of claim10, wherein the pharmaceutical composition is administered at leasttwice a day.
 12. A method of preventing lymphedema, the methodcomprising administering to a subject at risk of or susceptible todeveloping lymphedema a pharmaceutical composition comprising aneffective amount of pirfenidone, wherein the pharmaceutical compositionis administered prophylactically within about six weeks of a lymphaticinjury.
 13. The method of claim 12, wherein the pharmaceuticalcomposition is administered prophylactically within about two weeks of alymphatic injury.
 14. The method of claim 12, wherein the pharmaceuticalcomposition is administered topically.
 15. The method of claim 14,wherein the pharmaceutical composition is in a form selected from thegroup consisting of an ointment, a cream, a lotion, a paste, a gel, amousse, a foam, a lacquer, a suspension, a liquid, and a spray.
 16. Themethod of claim 15, wherein the pharmaceutical composition is in theform of an ointment.
 17. The method of claim 14, wherein thepharmaceutical composition is administered at least once a day.
 18. Themethod of claim 17, wherein the pharmaceutical composition isadministered at least twice a day.
 19. The method of claim 12, whereinthe pharmaceutical composition is administered orally.
 20. The method ofclaim 19, wherein the pharmaceutical composition is in a form selectedfrom the group consisting of a tablet, a capsule, a liquid, a solution,a softgel, a suspension, an emulsion, a syrup, and an elixir.
 21. Themethod of claim 20, wherein the pharmaceutical composition is in theform of a tablet or a capsule.
 22. The method of claim 19, wherein thepharmaceutical composition is administered at least once a day.
 23. Themethod of claim 22, wherein the pharmaceutical composition isadministered at least twice a day.